Stimulus-inducible protein kinase complex and methods of use therefor

ABSTRACT

Compositions and methods are provided for treating NF-κB-related conditions. In particular, the invention provides a stimulus-inducible IKK signalsome, and components and variants thereof. An IKK signalsome or component thereof may be used, for example, to identify antibodies and other modulating agents that inhibit or activate signal transduction via the NF-κB cascade. IKK signalsome, components thereof and/or modulating agents may also be used for the treatment of diseases associated with NF-κB activation.

CROSS-REFERENCE TO PRIOR APPLICATION

[0001] This is a divisional application of Ser. No. 08/910,820, filedAug. 17, 1997 which is a continuation-in-part of U.S. patent applicationSer. No. 08/697,393, filed Aug. 26, 1996.

TECHNICAL FIELD

[0002] The present invention relates generally to compositions andmethods useful for the study of cascades leading to the activation ofnuclear factor κB (NF-κB) and for treating diseases associated with suchpathways. The invention is more particularly related to astimulus-inducible IκB kinase (IKK) signalsome, component IκB kinasesand variants of such kinases. The present invention is also related tothe use of a stimulus-inducible IKK signalsome or IκB kinase to identifyantibodies and other agents that inhibit or activate signal transductionvia the NF-κB pathway.

BACKGROUND OF THE INVENTION

[0003] Transcription factors of the NFκB/Rel family are criticalregulators of genes involved in inflammation, cell proliferation andapoptosis (for reviews, see Verma et al., Genes Dev. 9:2723-35, 1995;Siebenlist, Biochim. Biophys. Acta 1332:7-13, 1997, Baeuerle and Henkel,Ann. Rev. Immunol. 12:141-79, 1994; Barnes and Karin, New Engl. J. Med.336,-1066-71, 1997; Baeuerle and Baltimore, Cell 87:13-20, 1996; Grilliet al., NF-κB and Rel: Participants in a multiform transcriptionalregulatory system (Academic Press, Inc., 1993), vol. 143; Baichwal andBaeuerle, Curr. Biol. 7:94-96, 1997). The prototype member of thefamily, NFκB, is composed of a dimer of p50 NFκB and p65 RelA (Baeuerleand Baltimore, Cell 53:211-17, 1988; Baeuerle and Baltimore, Genes Dev.3:1689-98, 1989). NF-κB plays a pivotal role in the highly specificpattern of gene expression observed for immune, inflammatory and acutephase response genes, including interleukin 1, interleukin 8, tumornecrosis factor and certain cell adhesion molecules.

[0004] Like other members of the Rel family of transcriptionalactivators, NF-κB is sequestered in an inactive form in the cytoplasm ofmost cell types. A variety of extracellular stimuli including mitogens,cytokines, antigens, stress inducing agents, UV light and viral proteinsinitiate a signal transduction pathway that ultimately leads to NF-κBrelease and activation. Thus, inhibitors and activators of the signaltransduction pathway may be used to alter the level of active NF-κB, andhave potential utility in the treatment of diseases associated withNF-κB activation.

[0005] Activation of NFκB in response to each of these stimuli iscontrolled by an inhibitory subunit, IκB, which retains NFκB in thecytoplasm. IκB proteins, of which there are six known members, eachcontain 5-7 ankyrin-like repeats required for association with theNFκB/Rel dimer and for inhibitory activity (see Beg et al., Genes Dev.7, 2064-70, 1993; Gilmore and Morin, Trends Genet. 9, 427-33, 1993;Diaz-Meco et al., Mol. Cell. Biol. 13:4770-75, 1993; Haskill et al.,Cell 65:1281-89, 1991). IκB proteins include IκBα and IκBβ.

[0006] NFκB activation involves the sequential phosphorylation,ubiquitination, and degradation of IκB. Phosphorylation of IκB is highlyspecific for target residues. For example, phosphorylation of the IκBprotein IκBα takes place at serine residues S32 and S36, andphosphorylation of IκBβ occurs at serine residues S19 and S23. Thechoreographed series of modification and degradation steps results innuclear import of transcriptionally active NFκB due to the exposure of anuclear localization signal on NFκB that was previously masked by IκB(Beg et al., Genes Dev. 6:1899-1913, 1992). Thus, NFκB activation ismediated by a signal transduction cascade that includes one or morespecific IκB kinases, a linked series of E1, E2 and E3 ubiquitinenzymes, the 26S proteasome, and the nuclear import machinery. Thephosphorylation of IκB is a critical step in NF-κB activation, and theidentification of an IκB kinase, as well as proteins that modulate itskinase activity, would further the understanding of the activationprocess, as well as the development of therapeutic methods.

[0007] Several protein kinases have been found to phosphorylate IκB invitro, including protein kinase A (Ghosh and Baltimore, Nature344:678-82, 1990), protein kinase C (Ghosh and Baltimore, Nature344:678-82, 1990) and double stranded RNA-dependent protein kinase(Kunar et al., Proc. Natl. Acad. Sci. USA 91:6288-92, 1994).Constitutive phosphorylation of IκBα by casein kinase II has also beenobserved (see Barroga et al., Proc. Natl. Acad. Sci. USA 92:7637-41,1995). None of these kinases, however appear to be responsible for invivo activation of NF-κB. For example, phosphorylation of IκBα in vitroby protein kinase A and protein kinase C prevent its association withNF-κB, and phosphorylation by double-stranded RNA-dependent proteinkinase results in dissociation of NF-κB. Neither of these conform to theeffect of phosphorylation in vivo, where IκBα phosphorylation at S32 andS36 does not result in dissociation from NF-κB.

[0008] Other previously unknown proteins with IκB kinase activity havebeen reported, but these proteins also do not appear to be significantactivators in vivo. A putative IκBα kinase was identified by Kuno etal., J. Biol. Chem. 270:27914-27919, 1995, but that kinase appears tophosphorylate residues in the C-terminal region of IκBα, rather than theS32 and S36 residues known to be important for in vivo regulation.Diaz-Meco et al., EMBO J. 13:2842-2848, 1994 also identified a 50 kD IκBkinase, with uncharacterized phosphorylation sites. Schouten et al.,EMBO J. 16:3133-44, 1997 identified p90^(rski) as a putative IκBαkinase; however, p90^(rski) is only activated by TPA and phosphorylatesIκBα only on Ser32, which is insufficient to render IκBα a target forubiquitination. Finally, Chen et al, Cell 84:853-862, 1996 identified akinase that phosphorylates IκBα, but that kinase was identified using anon-physiological inducer of IκBα kinase activity and requires theaddition of exogenous factors for in vitro phosphorylation.

[0009] Accordingly, there is a need in the art for an IκB kinase thatpossesses the substrate specificity and other properties of the in vivokinase. There is also a need for improved methods for modulating theactivity of proteins involved in activation of NF-κB, and for treatingdiseases associated with NF-κB activation. The present inventionfulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

[0010] Briefly stated, the present invention provides compositions andmethods employing a large, multi-subunit IKK signalsome, or a componentor variant thereof. In one aspect, the present invention provides an IKKsignalsome capable of specifically phosphorylating IκBα at residues S32and S36, and IκBβ at residues 19 and 23, without the addition ofexogenous cofactors.

[0011] In a further related aspect, a polypeptide comprising a componentof an IKK signalsome, or a variant of such a component, is provided,wherein the component has a sequence recited in SEQ ID NO:9. An isolatedDNA molecule and recombinant expression vector encoding such apolypeptide, as well as a transfected host cell, are also provided.

[0012] In another aspect, methods for preparing an IKK signalsome areprovided, comprising combining components of an IKK signalsome in asuitable buffer.

[0013] In yet another aspect, methods are provided for phosphorylating asubstrate of an IKK signalsome, comprising contacting a substrate withan IKK signalsome or a component thereof, and thereby phosphorylatingthe substrate.

[0014] In a further aspect, the present invention provides a method forscreening for an agent that modulates IKK signalsome activity,comprising: (a) contacting a candidate agent with an IKK signalsome,wherein the step of contacting is carried out under conditions and for atime sufficient to allow the candidate agent and the IKK signalsome tointeract; and (b) subsequently measuring the ability of the candidateagent to modulate IKK signalsome activity.

[0015] Within a related aspect, the present invention provides methodsfor screening for an agent that modulates IKK signalsome activity,comprising: (a) contacting a candidate agent with a polypeptidecomprising a component of an IKK signalsome as described above, whereinthe step of contacting is carried out under conditions and for a timesufficient to allow the candidate agent and the polypeptide to interact;and (b) subsequently measuring the ability of the candidate agent tomodulate the ability of the polypeptide to phosphorylate an IκB protein.

[0016] In another aspect, an antibody is provided that binds to acomponent (e.g., IKK-1 and/or IKK-2) of an IKK signalsome, where thecomponent is capable of phosphorylating IκBα.

[0017] In further aspects, the present invention provides methods formodulating NF-κB activity in a patient, comprising administering to apatient an agent that modulates IκB kinase activity in combination witha pharmaceutically acceptable carrier. Methods are also provided fortreating a patient afflicted with a disorder associated with theactivation of IKK signalsome, comprising administering to a patient atherapeutically effective amount of an agent that modulates IκB kinaseactivity in combination with a pharmaceutically acceptable carrier.

[0018] In yet another aspect, a method for detecting IKK signalsomeactivity in a sample is provided, comprising: (a) contacting a samplewith an antibody that binds to an IKK signalsome under conditions andfor a time sufficient to allow the antibody to immunoprecipitate an IKKsignalsome; (b) separating immunoprecipitated material from the sample;and (c) determining the ability of the immunoprecipitated material tospecifically phosphorylate an IκB protein with in vivo specificity.Within one such embodiment, the ability of the immunoprecipitatedmaterial to phosphorylate IκBα at residues S32 and/or S36 is determined.

[0019] In a related aspect, a kit for detecting IKK signalsome activityin a sample is provided, comprising an antibody that binds to an IKKsignalsome in combination with a suitable buffer.

[0020] In a further aspect, the present invention provides a method foridentifying an upstream kinase in the NF-κB signal transduction cascade,comprising evaluating the ability of a candidate upstream kinase tophosphorylate an IKK signalsome, a component thereof or a variant ofsuch a component.

[0021] A method for identifying a component of an IKK signalsome is alsoprovided, comprising: (a) isolating an IKK signalsome; (b) separatingthe signalsome into components, and (c) obtaining a partial sequence ofa component.

[0022] In yet another aspect, a method is provided for preparing an IKKsignalsome from a biological sample, comprising: (a) separating abiological sample into two or more fractions; and (b) monitoring IκBkinase activity in the fractions.

[0023] These and other aspects of the present invention will becomeapparent upon reference to the following detailed description andattached drawings. All references disclosed herein are herebyincorporated by reference in their entirety as if each was incorporatedindividually.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIGS. 1A-1C are autoradiograms depicting the results ofimmunoblot analyses. FIG. 1A shows the recruitment of IκBα into a highmolecular weight complex upon stimulation. Cytoplasmic extracts ofeither unstimulated or PMA(50 ng/ml)- and PHA(1 μg/ml)-stimulated (10min) Jurkat cells were fractionated on a gel filtration column. IκBα wasvisualized by immunoblot analysis. The upper panel shows the elutionprofile of unstimulated cells, and the lower panel shows the elutionprofile of PMA/PHA-stimulated cells. Molecular weight standards areindicated by arrows on the top.

[0025]FIG. 1B shows that the stimulus-dependent IκBα kinase activitychromatographs as a high molecular weight complex, M_(T) 500-700 kDa.Whole cell extract of TNFα-stimulated (20 ng/ml, 7 min) HeLa S3 cellswas fractionated on a Superdex 200 gel filtration column and monitoredfor IκBα kinase activity. Phosphorylation of the GST IκBα 1-54(wildtype) substrate is indicated by an arrow to the right. Molecularweight standards are indicated by arrows on the top.

[0026]FIG. 1C illustrates the identification of proteins thatco-chromatograph with the IKK signalsome. IKK signalsome was partiallypurified from extracts of TNFα-stimulated HeLa S3 cells by sequentialfractionation on a Q Sepharose, Superdex 200, Mono Q, and PhenylSuperose columns. Phenyl Superose fractions containing the peak of IKKsignalsome activity were subjected to western blot analysis usingseveral different antibodies as indicated to the left of each respectivepanel. The level of IKK signalsome activity is indicated in the uppershaded area by increasing number of (+)'s.

[0027]FIG. 2 is a flow chart depicting a representative purificationprocedure for the preparation of an IKK signalsome.

[0028]FIGS. 3A and 3B are autoradiograms that show the results of aWestern blot analysis of the levels of IκBα in HeLa S3 cytoplasmicextracts following gel filtration. The extracts were prepared from cellsthat were (FIG. 3B) and were not (FIG. 3A) exposed to TNFα.

[0029]FIGS. 4A and 4B are autoradiograms depicting the results of an invitro kinase assay in which the ability of the above cell extracts tophosphorylate the N-terminal portion of IκBα was evaluated. FIG. 4Ashows the results employing an extract from cells that were not treatedwith TNFα, and FIG. 4B shows the results when the cells were treatedwith TNFα.

[0030]FIGS. 5A and 5B are autoradiograms depicting the results of an invitro kinase assay using a cytoplasmic extract of TNFα-treated HeLa S3cells, where the extract is subjected to Q Sepharose fractionation. Thesubstrate was the truncated IκBα (residues 1 to 54), with FIG. 5Ashowing the results obtained with the wild type IκBα sequence and FIG.5B presenting the results obtained using a polypeptide containingthreonine substitutions at positions 32 and 36.

[0031]FIGS. 6A and 6B are autoradiograms depicting the results of an invitro kinase assay using a cytoplasmic extract of TNFα-treated HeLa S3cells, where the extract was subjected in series to chromatographicfractionation by Q Sepharose, Superdex 200, Mono Q Sepharose and PhenylSuperose. The substrate was the truncated IκBα (residues 1 to 54), withFIG. 6A showing the results obtained with the wild type IκBα sequenceand FIG. 6B presenting the results obtained using a polypeptidecontaining threonine substitutions at positions 32 and 36.

[0032]FIG. 7 is an autoradiogram showing the results of immunokinaseassays (using anti-MKP-1 antibody) performed using cytoplasmic extractsof TNFα-treated HeLa S3 cells following gel filtration. The assay wasperformed using the substrates GST-IκBα1-54 wildtype (lane 1) andGST-IκBα1-54 S32/36 to T (lane 2). The positions of IκBα and GST-IκBα1-54 are indicated on the left.

[0033] FIGS. 8A-8C are autoradiograms depicting the results ofimmunoblot analyses. In FIG. 8A, the upper panel presents a time coursefor the induction of signalsome activity. Anti MKP-1 immune precipitatesfrom extracts of HeLa S3 cells stimulated with TNFα (20 ng/ml) for theindicated times, were assayed for IKK signalsome activity by standardimmune complex kinase assays. 4 μg of either GST IκBα 1-54 WT (wildtype)or the GST IκBα 1-54 S32/36 to T mutant (S>T) were used as thesubstrates. In the lower panel, HeLa cell extracts prepared as describedin the upper panel were examined by western blot analysis for IκBαdegradation. IκBα supershifting phosphorylation can be seen after 3 and5 minutes of stimulation followed by the disappearance of IκBα.

[0034]FIG. 8B illustrates the stimulus-dependent activation of IKKsignalsome, which is blocked by TPCK. Anti-MKP-1 immunoprecipitates fromcell extracts of HeLa S3 cells either stimulated for 7 min with TNFα (20ng/ml, lane 2 and 6), IL-1 (10 ng/ml, lane 3), PMA (50 ng/ml, lane 4) orpretreated for 30 min with TPCK (15 μM, lane 7) prior to TNFα-inductionwere examined for IKK signalsome activity. GST IκBα 1-54 WT (4 μg) wasused as a substrate.

[0035]FIG. 8C illustrates the ability of IKK signalsome to specificallyphosphorylate serines 32 and 36 of the IκBα holoprotein in the contextof a RelA:IκBα complex. Anti-MKP-1 immunoprecipitates from cell extractsof HeLa S3 cells stimulated with TNFα (20 ng/ml, 7 min) were examinedfor their ability to phosphorylate baculoviral expressed RelA:IκBαcomplex containing either the IκBα WT (lane 3) or IκBα S32/36 to Amutant (lane 4) holoprotein. The specific substrates used are indicatedon the top. Positions of the phosphorylated substrates are indicated byarrows to the left of the panel.

[0036]FIG. 9A is an autoradiogram depicting the results of animmunokinase assay in which peptides are phosphorylated by the IKKsignalsome. In the top panel, IκBα (21-41) peptides that wereunphosphorylated or chemically phosphorylated on either Ser-32 or Ser-36were incubated with the IKK signalsome in the presence of γ-[³²P]-ATP.The doubly phosphorylated peptide P32,36 was not phosphorylated by theIKK signalsome, and the unrelated c-Fos(222-241) phosphopeptide withfree serine and threonine residues did not function as a signalsomesubstrate.

[0037]FIG. 9B is a graph illustrating the inhibition of phosphorylationof GST-IκBα (1-54) by IκBα (21-41) peptides. IκBα (21-41) peptide P32,36inhibits GST-IκBα (1-54) as a product inhibitor with a K_(i) value of 14μM. The unrelated phosphopeptide c-Fos(222-241) does not function as aninhibitor. This assay only detects precipitated ³²P-labeled proteins,not ³²P-labeled peptides. Addition of the singly- or non-phosphorylatedIκBα (21-41) peptides results in less phosphorylation of GST-IκBα (1-54)and apparent inhibition.

[0038]FIG. 10 is an autoradiogram showing the results of a western blotanalysis of the level of ubiquitin within a stimulus-inducible IkBkinase complex. Lane 1 shows the detection of 100 ng ubiquitin, Lane 2shows 10 ng ubiquitin and Lane 3 shows 3.4 μg of IKK signalsome purifiedthrough the phenyl superose step (sufficient quantities for 10 kinasereactions). The position of ubiquitin is shown by the arrow on the left.

[0039]FIG. 11A illustrates a procedure for purification of the IKKsignalsome. A whole cell extract was prepared from TNFα-stimulated (20ng/ml, 7 minute induction) HeLa S3 cells (1.2 g total protein). The IKKsignalsome was then immunoprecipitated from the extract using anti-MKP-1antibodies, washed with buffer containing 3.5 M urea and elutedovernight at 4° C. in the presence of excess MKP-1 specific peptide.Eluted IKK signalsome was then fractionated on a Mono Q column, IκBkinase active fractions were pooled, concentrated and subjected topreparative SDS-PAGE. Individual protein bands were excised andsubmitted for peptide sequencing.

[0040]FIG. 11B is a photograph showing Mono Q fractions containingactive IKK signalsome activity following SDS-PAGE and a standard silverstain protocol. Peak activity of IKK signalsome activity is representedin lanes 3, 4, and 5. Protein bands corresponding to IKK-1 and IKK-2 areindicated to the left of the figure. Molecular weight standards (kDa)are indicated to the left of the figure.

[0041]FIGS. 12A and 12B are mass spectra obtained during sequencing ofIKK-2 by nanoelectrospray mass spectrometry. FIG. 12A shows part of themass spectrum of the unseparated mixture of tryptic peptides resultingfrom in-gel digestion of the IKK-2 band in FIG. 11B. FIG. 12B shows atandem mass spectrum of the peak at m/z 645.2.

[0042]FIG. 13A illustrates the amino acid sequence of IKK-1 and IKK-2.Symbols: arrows, boundaries of the kinase domain; underlined, peptidesequences identified by nanoelectrospray mass spectrometry; asterisks,indicates leucines comprising the leucine zipper motif; bold face,indicate amino acid identities conserved between IKK-1 and IKK-2;highlighted box, Helix-loop-helix domain; dashes, a gap inserted tooptimize alignment.

[0043]FIG. 13B is an autoradiogram depicting the results of Northernblot analysis of IKK-2 mRNA in adult human tissue. The source of thetissue is indicated at the top. Probes spanning the coding region ofhuman IKK-2 and β-actin cDNA were used and are indicated to the left.Molecular weight standards are indicated to the right.

[0044]FIG. 14A is an autoradiogram depicting the results of kinaseassays using IKK-1 and IKK-2. IKK-1 and IKK-2 were immunoprecipitatedfrom rabbit reticulocyte lysates phosphorylate Iκα and IκBβ. EitherHA-tagged IKK-1 (lane 1) or Flag-tagged IKK-2 (lane 2) were translatedin rabbit reticulocyte lysates, immunoprecipitated, and examined fortheir ability to phosphorylate GST IκBα 1-54 WT and GST IκBβ 1-44 asindicated by an arrow to the left. IKK-1 (lane 1) undergoes significantautophosphorylation in contrast to IKK-2 (lane 2) which is identifiedonly with longer exposure times.

[0045]FIGS. 14B and 14C are micrographs illustrating the results ofassays to evaluate the ability of kinase-inactive mutants of IKK-1 andIKK-2 to inhibit RelA translocation in TNFα-stimulated HeLa cells. HeLacells were transiently transfected with either HA-tagged IKK-1 K44 to Mmutant (14B) or Flag-tagged IKK-2 K44 to M mutant (14C) expressionvectors. 36 hours post-transfection cells were either not stimulated(Unstim) or TNFα-stimulated (20 ng/ml) for 30 min (TNFα), as indicatedto the right of the figure. Cells were then subjected toimmunofluorescence staining using anti-HA of anti-Flag antibodies tovisualize expression of IKK-1 K44 to M or IKK-2 K44 to M, respectively.Stimulus-dependent translocation of Rel A was monitored using anti-Rel Aantibodies. Antibodies used are indicated to the top of the figure. IKKmutant transfected is indicated to the left of the figure.

[0046]FIGS. 15A and 15B are autoradiograms of immunoprecipitated IKK-1and IKK-2 following in vitro translation. In FIG. 15A, HA-tagged IKK-1and Flag-tagged IKK-2 were in vitro translated in wheat germ lysateseither separately or in combination, as indicated. The programmedtranslation mix was then subjected to immunoprecipitation using theindicated antibody. The samples were run on SDS-PAGE and subjected toautoradiography. In FIG. 15B, HA-tagged IKK-1 and Flag-tagged IKK-2 werein vitro translated in rabbit reticulocyte lysates either separately orin combination, as indicated. The programmed translation mix was thensubjected to immunoprecipitation using the indicated antibody. Thesamples were run on SDS-PAGE and subjected to autoradiography. Theresults show that IKK-1 and IKK-2 coprecipitate when translated inrabbit reticulocyte lysates.

DETAILED DESCRIPTION OF THE INVENTION

[0047] As noted above, the present invention is generally directed tocompositions and methods for modulating (i.e., stimulating orinhibiting) signal transduction leading to NF-κB activation. Inparticular, the present invention is directed to compositions comprisingan IκB kinase (IKK) signalsome (also referred to herein as a“stimulus-inducible IκB kinase complex” or “IκB kinase complex”) that iscapable of stimulus-dependent phosphorylation of IκBα and IκBβ on thetwo N-terminal serine residues critical for the subsequentubiquitination and degradation in vivo. Such stimulus-dependentphosphorylation may be achieved without the addition of exogenouscofactors. In particular, an IKK signalsome specifically phosphorylatesIκBα (SEQ ID NO:1) at residues S32 and S36 and phosphorylates IκBβ (SEQID NO:2) at residues S19 and S23. The present invention also encompassescompositions that contain one or more components of such an IKKsignalsome, or variants of such components. Preferred components,referred to herein as “IKK signalsome kinases” “IκB kinases” or IKKs)are kinases that, when incorporated into an IKK signalsome, are capableof phosphorylating IκBα at S32 and S36. Particularly preferredcomponents are IKK-1 (SEQ ID NO:10) and IKK-2 (SEQ ID NO:9).

[0048] An IKK signalsome and/or IκB kinase may generally be used forphosphorylating a substrate (i e., an IκB, such as IκBα, or a portion orvariant thereof that can be phosphorylated at those residues that arephosphorylated in vivo) and for identifying modulators of IκB kinaseactivity. Such modulators and methods employing them for modulating IκBαkinase activity, in vivo and/or in vitro, are also encompassed by thepresent invention. In general, compositions that inhibit IκB kinaseactivity may inhibit IκB phosphorylation, or may inhibit the activationof an IκB kinase and/or IKK signalsome.

[0049] An IKK signalsome has several distinctive properties. Such acomplex is stable (i.e., its components remain associated followingpurification as described herein) and has a high-molecular weight (about500-700 kD, as determined by gel filtration chromatography). As shown inFIGS. 3(A and B) and 4(A and B), IκB kinase activity of an IKKsignalsome is “stimulus-inducible” in that it is stimulated by TNFα(i.e., treatment of cells with TNFα results in increased IκB kinaseactivity and IκB degradation) and/or by one or more other inducers ofNF-κB, such as IL-1, LPS, TPA, UV irradiation, antigens, viral proteinsand stress-inducing agents. The kinetics of stimulation by TNFαcorrespond to those found in vivo. IκB kinase activity of an IKKsignalsome is also specific for S32 and S36 of IκBα. As shown in FIGS.5(A and B) and 6(A and B), an IKK signalsome is capable ofphosphorylating a polypeptide having the N-terminal sequence of IκBα(GST-IκBα1-54; SEQ ID NO:3), but such phosphorylation cannot be detectedin an IκBα derivative containing threonine substitutions at positions 32and 36. In addition, IκB kinase activity is strongly inhibited by adoubly phosphorylated IκBα peptide (i.e., phosphorylated at S32 andS36), but not by an unrelated c-fos phosphopeptide that contains asingle phosphothreonine. A further characteristic of an IKK signalsomeis its ability to phosphorylate a substrate in vitro in a standardkinase buffer, without the addition of exogenous cofactors. Freeubiquitin is not detectable in preparations of IKK signalsome (see FIG.10), even at very long exposures. Accordingly an IKK signalsome differsfrom the ubiquitin-dependent IκBα kinase activity described by Chen etal., Cell 84:853-62, 1996.

[0050] An IKK signalsome may be immunoprecipitated by antibodies raisedagainst MKP-1 (MAP kinase phosphatase-1; Santa Cruz Biotechnology, Inc.,Santa Cruz, Calif. #SC-1102), and its activity detected using an invitro IκBα kinase assay. However, as discussed further below, MKP-1 doesnot appear to be a component of IκB kinase complex. The substratespecificity of the immunoprecipitated IKK signalsome is maintained(i.e., there is strong phosphorylation of wildtype GST-IκBα 1-54 (SEQ IDNO:3) and GST-IκBβ 1-44 (SEQ ID NO:4), and substantially no detectablephosphorylation of GST-IκBα 1-54 in which serines 32 and 36 are replacedby threonines (GST-IκBα S32/36 to T; SEQ ID NO:5) or GST-IκBβ 1-44 inwhich serines 19 and 23 are replaced by alanines (GST-IκBβ 1-44 S19/23to A; SEQ ID NO:6)).

[0051] An IKK signalsome may be isolated from human or other cells, andfrom any of a variety of tissues and/or cell types. For example, usingstandard protocols, cytoplasmic and/or nuclear/membrane extracts may beprepared from HeLa S3 cells following seven minutes induction with 30ng/mL TNFα. The extracts may then be subjected to a series ofchromatographic steps that includes Q Sepharose, gel filtration (HiLoad16/60 Superdex 200), Mono Q, Phenyl Superose, gel filtration (Superdex200 10/30) and Mono Q. This representative purification procedure isillustrated in FIG. 2, and results in highly enriched IKK signalsome(compare, for example, FIGS. 5A and 6A).

[0052] An alternative purification procedure employs a two-step affinitymethod, based on recognition of IKK signalsome by the MKP-1 antibody(FIG. 11A). Whole cell lysates from TNFα-stimulated HeLa cells may beimmunoprecipitated with an anti-MKP-1 antibody. The IKK signalsome maybe eluted with the specific MKP-1 peptide to which the antibody wasgenerated and fractionated further on a Mono Q column.

[0053] Throughout the fractionation, an in vitro kinase assay may beused to monitor the IκB kinase activity of the IKK signalsome. In suchan assay, the ability of a fraction to phosphorylate an appropriatesubstrate (such as IκBα (SEQ ID NO:1) or a derivative or variantthereof) is evaluated by any of a variety of means that will be apparentto those of ordinary skill in the art. For example, a substrate may becombined with a chromatographic fraction in a protein kinase buffercontaining ³²P γ-ATP, phosphatase inhibitors and protease inhibitors.The mixture may be incubated for 30 minutes at 30° C. The reaction maythen be stopped by the addition of SDS sample buffer and analyzed usingSDS-PAGE with subsequent autoradiography. Suitable substrates includefull length IκBα (SEQ ID NO: 1), polypeptides comprising the N-terminal54 amino acids of IκBα, full length IκBβ (SEQ ID NO:2) and polypeptidescomprising the N-terminal 44 amino acids of IκBβ. Any of thesesubstrates may be used with or without an N-terminal tag. One suitablesubstrate is a protein containing residues 1-54 of IκBα and anN-terminal GST tag (referred to herein as GST-IκBα 1-54; SEQ ID NO:3).To evaluate the specificity of an IκB kinase complex, IκBα mutantscontaining threonine or alanine residues at positions 32 and 36, and/orother modifications, may be employed.

[0054] Alternatively, an IKK signalsome may be prepared from itscomponents which are also encompassed by the present invention. Suchcomponents may be produced using well known recombinant techniques, asdescribed in greater detail below. Components of an IKK signalsome maybe native, or may be variants of a native component (i.e., a componentsequence may differ from the native sequence in one or moresubstitutions and/or modifications, provided that the ability of acomplex comprising the component variant to specifically phosphorylateIκBα is not substantially diminished). Substitutions and/ormodifications may generally be made in non-critical and/or criticalregions of the native protein. Variants may generally comprise residuesof L-amino acids, D-amino acids, or any combination thereof. Amino acidsmay be naturally-occuring or may be non-natural, provided that at leastone amino group and at least one carboxyl group are present in themolecule; α- and β-amino acids are generally preferred. A variant mayalso contain one or more rare amino acids (such as 4-hydroxyproline orhydroxylysine), organic acids or amides and/or derivatives of commonamino acids, such as amino acids having the C-terminal carboxylateesterified (e.g., benzyl, methyl or ethyl ester) or amidated and/orhaving modifications of the N-terminal amino group (e.g., acetylation oralkoxycarbonylation), with or without any of a wide variety ofside-chain modifications and/or substitutions (e.g., methylation,benzylation, t-butylation, tosylation, alkoxycarbonylation, and thelike). Component variants may also, or alternatively, contain othermodifications, including the deletion or addition of amino acids thathave minimal influence on the activity of the polypeptide. Inparticular, variants may contain additional amino acid sequences at theamino and/or carboxy termini. Such sequences may be used, for example,to facilitate purification or detection of the component polypeptide. Ingeneral, the effect of one or more substitutions and/or modificationsmay be evaluated using the representative assays provided herein.

[0055] A component may generally be prepared from a DNA sequence thatencodes the component using well known recombinant methods. DNAsequences encoding components of an IKK signalsome may be isolated by,for example, screening a suitable expression library (i.e., a libraryprepared from a cell line or tissue that expresses IKK signalsome, suchas spleen, leukocytes, HeLa cells or Jurkat cells) with antibodiesraised against IKK signalsome or against one or more components thereof.Protein components may then be prepared by expression of the identifiedDNA sequences, using well known recombinant techniques.

[0056] Alternatively, partial sequences of the components may beobtained using standard biochemical purification and microsequencingtechniques. For example, purified complex as described above may be runon an SDS-PAGE gel and individual bands may be isolated and subjected toprotein microsequencing. DNA sequences encoding components may then beprepared by amplification from a suitable human cDNA library, usingpolymerase chain reaction (PCR) and methods well known to those ofordinary skill in the art. For example, an adapter-ligated cDNA libraryprepared from a cell line or tissue that expresses IKK signalsome (suchas HeLa or Jurkat cells) may be screened using a degenerate 5′ specificforward primer and an adapter-specific primer. Degenerateoligonucleotides may also be used to screen a cDNA library, usingmethods well known to those of ordinary skill in the art. In addition,known proteins may be identified via Western blot analysis usingspecific antibodies.

[0057] Components of an IKK signalsome may also be identified byperforming any of a variety of protein-protein interaction assays knownto those of ordinary skill in the art. For example a known component canbe used as “bait” in standard two-hybrid screens to identify otherregulatory molecules, which may include IKK-1, IKK-2, NFκB1, RelA, IκBβand/or p70 S6 kinase (Kieran et al., Cell 62:1007-1018, 1990; Nolan etal., Cell 64:961-69, 1991; Thompson et al., Cell 80:573-82, 1995; Groveet al., Mol. Cell Biol. 11:5541-50, 1991).

[0058] Particularly preferred components of IKK signalsome are IκBkinases. An IκB kinase may be identified based upon its ability tophosphorylate one or more IκB proteins, which may be readily determinedusing the representative kinase assays described herein. In general, anIκB kinase is incorporated into an IKK signalsome, as described herein,prior to performing such assays, since an IκB kinase that is notcomplex-associated may not display the same phosphorylation activity ascomplex-associated IκB kinase. As noted above, an IκB kinase within anIKK signalsome specifically phosphorylates IκBα at residues S32 and S36,and phosphorylates IκBβ at residues 19 and 23, in response to specificstimuli.

[0059] As noted above, IKK-1 and IKK-2 are particularly preferred IκBkinases. IKK-1 and IKK-2 may be prepared by pooling the fractions fromthe Mono Q column containing peak IκB kinase activity and subjecting thepooled fractions to preparative SDS gel electrophoresis. The intensityof two prominent protein bands of ˜85 and ˜87 kDa (indicated by silverstain in FIG. 11B as IKK-1 and IKK-2 respectively) correlates with theprofile of IκB kinase activity. The ˜85 kDa band, corresponding toIKK-1, has been identified, within the context of the present invention,as CHUK (conserved helix-loop-helix ubiquitous kinase; see Connely andMarcu, Cell. Mol. Biol. Res. 41:537-49,1995). The ˜87 kDa band containsIKK-2.

[0060] Sequence analysis reveals that IKK-1 and IKK-2 are relatedprotein serine kinases (51% identity) containing protein interactionmotifs (FIG. 13A). Both IKK-1 and IKK-2 contain the kinase domain at theN-terminus, and a leucine zipper motif and a helix-loop-helix motif intheir C-terminal regions. Northern analysis indicates that mRNAsencoding IKK-2 are widely distributed in human tissues, with transcriptsizes of ˜4.5 kb and 6 kb (FIG. 13B). The sequences of IKK-1 and IKK-2are also provided as SEQ ID NOs: 7 and 8, respectively.

[0061] It has been found, within the context of the present invention,that rabbit reticulocyte lysate immunoprecipitates that contain IKK-1 orIKK-2 phosphorylate IκBα and IκBβ with the correct substrate specificity(see FIG. 14A). Altered versions of these kinases interfere withtranslocation of RelA to the nucleus of TNFα-stimulated HeLa cells.Accordingly, IKK-1 and IKK-2 appear to control a significant early stepof NFκB activation.

[0062] Other components of an IKK signalsome are also contemplated bythe present invention. Such components may include, but are not limitedto, upstream kinases such as MEKK-1 (Lee et al., Cell 88,:213-22, 1997;Hirano et al., J. Biol. Chem. 271:13234-38, 1996) or NIK (Malinin etal., Nature 385:540-44, 1997); adapter proteins that mediate anIKK-1:IKK-2 interaction; a component that crossreacts with anti-MKP-1;an inducible RelA kinase; and/or the E3 ubiquitin ligase that transfersmultiubiquitin chains to phosphorylated IκB (Hershko and Ciechanover,Annu. Rev. Biochem. 61:761-807, 1992).

[0063] A component of an IKK signalsome may generally be prepared fromDNA encoding the component by expression of the DNA in cultured hostcells, which may be stable cell lines or transiently transfected cells.Preferably, the host cells are bacteria, yeast, baculovirus-infectedinsect cells or mammalian cells. The recombinant DNA may be cloned intoany expression vector suitable for use within the host cell, usingtechniques well known to those of ordinary skill in the art. Anexpression vector may, but need not, include DNA encoding an epitope,such that the recombinant protein contains the epitope at the N- orC-terminus. Epitopes such as glutathione-S transferase protein (GST), HA(hemagglutinin)-tag, FLAG and Histidine-tag may be added usingtechniques well known to those of ordinary skill in the art.

[0064] The DNA sequences expressed in this manner may encode nativecomponents of an IKK signalsome, or may encode portions or variants ofnative components, as described above. DNA molecules encoding variantsmay generally be prepared using standard mutagenesis techniques, such asoligonucleotide-directed site-specific mutagenesis. Sections of the DNAsequence may also, or alternatively, be removed to permit preparation oftruncated polypeptides and DNA encoding additional sequences such as“tags” may be added to the 5′ or 3′ end of the DNA molecule.

[0065] IKK signalsome components may generally be used to reconstituteIKK signalsome. Such reconstitution may be achieved in vitro bycombining IKK signalsome components in a suitable buffer. Alternatively,reconstitution may be achieved in vivo by expressing components in asuitable host cell, such as HeLa or HUVEC, as described herein.

[0066] Expressed IKK signalsome, or a component thereof, may be isolatedin substantially pure form. Preferably, IKK signalsome or a component isisolated to a purity of at least 80% by weight, more preferably to apurity of at least 95% by weight, and most preferably to a purity of atleast 99% by weight. In general, such purification may be achievedusing, for example, the representative purification methods describedherein or the standard techniques of ammonium sulfate fractionation,SDS-PAGE electrophoresis, and affinity chromatography. IKK signalsomeand components for use in the methods of the present invention may benative, purified or recombinant.

[0067] In one aspect of the present invention, an IKK signalsome and/orone or more components thereof may be used to identify modulatingagents, which may be antibodies (e.g., monoclonal), polynucleotides orother drugs, that inhibit or stimulate signal transduction via the NF-κBcascade. Modulation includes the suppression or enhancement of NF-κBactivity. Modulation may also include suppression or enhancement of IκBphosphorylation or the stimulation or inhibition of the ability ofactivated (i.e., phosphorylated) IKK signalsome to phosphorylate asubstrate. Compositions that inhibit NF-κB activity by inhibiting IκBphosphorylation may include one or more agents that inhibit or blockIκBα kinase activity, such as an antibody that neutralizes IKKsignalsome, a competing peptide that represents the substrate bindingdomain of IκB kinase or a phosphorylation motif of IκB, an antisensepolynucleotide or ribozyme that interferes with transcription and/ortranslation of IκB kinase, a molecule that inactivates IKK signalsome bybinding to the complex, a molecule that binds to IκBα and preventsphosphorylation by IKK signalsome or a molecule that prevents transferof phosphate groups from the kinase to the substrate. Within certainembodiments, a modulating agent inhibits or enhances the expression oractivity of IKK-1 and/or IKK-2.

[0068] In general, modulating agents may be identified by combining atest compound with an IKK signalsome, IκB kinase or a polynucleotideencoding an IκB kinase in vitro or in vivo, and evaluating the effect ofthe test compound on the IκB kinase activity using, for example, arepresentative assay described herein. An increase or decrease in kinaseactivity can be measured by adding a radioactive compound, such as³²P-ATP and observing radioactive incorporation into a suitablesubstrate for IKK signalsome, thereby determining whether the compoundinhibits or stimulates kinase activity. Briefly, a candidate agent maybe included in a reaction mixture containing compounds necessary for thekinase reaction (as described herein) and IκB substrate, along with IKKsignalsome, IκB kinase or a polynucleotide encoding an IκB kinase. Ingeneral, a suitable amount of antibody or other agent for use in such anassay ranges from about 0.01 μM to about 10 μM. The effect of the agenton IκB kinase activity may then be evaluated by quantitating theincorporation of [³²P]phosphate into an IκB such as IκBα (or aderivative or variant thereof), and comparing the level of incorporationwith that achieved using IκB kinase without the addition of a candidateagent. Alternatively, the effect of a candidate modulating agent ontranscription of an IκB kinase may be measured, for example, by Northernblot analysis or a promoter/reporter-based whole cell assay.

[0069] Alternatively, for assays in which the test compound is combinedwith an IKK signalsome, the effect on a different IKK signalsomeactivity may be assayed. For example, an IKK signalsome also displaysp65 kinase activity and IKK phosphatase activity. Assays to evaluate theeffect of a test compound on such activities may be performed using wellknown techniques. For example, assays for p65 kinase activity maygenerally be performed as described by Zhong et al., Cell 89:413-24,1997. For phosphatase activity, an assay may generally be performed asdescribed by Sullivan et al., J. Biomolecular Screening 2:19-24, 1997,using a recombinant phosphorylated IκB kinase as a substrate.

[0070] In another aspect of the present invention, IKK signalsome or IκBkinase may be used for phosphorylating an IκB such as IκBα (or aderivative or variant thereof) so as to render it a target forubiquitination and subsequent degradation. IκB may be phosphorylated invitro by incubating IKK signalsome or IκB kinase with IκB in a suitablebuffer for 30 minutes at 30° C. In general, about 0.01 μg to about 9 μgof IκB kinase complex is sufficient to phosphorylate from about 0.5 μgto about 2 μg of IκB. Phosphorylated substrate may then be purified bybinding to GSH-sepharose and washing. The extent of substratephosphorylation may generally be monitored by adding [γ-³²P]ATP to atest aliquot, and evaluating the level of substrate phosphorylation asdescribed herein.

[0071] An IKK signalsome, component thereof, modulating agent and/orpolynucleotide encoding a component and/or modulating agent may also beused to modulate NF-κB activity in a patient. Such modulation may occurby any of a variety of mechanisms including, but not limited to, directinhibition or enhancement of IκB phosphorylation using a component ormodulating agent; or inhibiting upstream activators, such as NIK or MEK,with IKK signalsome or a component thereof. As used herein, a “patient”may be any mammal, including a human, and may be afflicted with adisease associated with IκB kinase activation and the NF-κB cascade, ormay be free of detectable disease. Accordingly, the treatment may be ofan existing disease or may be prophylactic. Diseases associated with theNF-κB cascade include inflammatory diseases, neurodegenerative diseases,autoimmune diseases, cancer and viral infection.

[0072] Treatment may include administration of an IKK signalsome, acomponent thereof and/or an agent which modulates IκB kinase activity.For administration to a patient, one or more such compounds aregenerally formulated as a pharmaceutical composition. A pharmaceuticalcomposition may be a sterile aqueous or non-aqueous solution, suspensionor emulsion, which additionally comprises a physiologically acceptablecarrier (i.e., a non-toxic material that does not interfere with theactivity of the active ingredient). Any suitable carrier known to thoseof ordinary skill in the art may be employed in the pharmaceuticalcompositions of the present invention. Representative carriers includephysiological saline solutions, gelatin, water, alcohols, natural orsynthetic oils, saccharide solutions, glycols, injectable organic esterssuch as ethyl oleate or a combination of such materials. Optionally, apharmaceutical composition may additionally contain preservatives and/orother additives such as, for example, antimicrobial agents,anti-oxidants, chelating agents and/or inert gases, and/or other activeingredients.

[0073] Alternatively, a pharmaceutical composition may comprise apolynucleotide encoding a component of an IKK signalsome and/or amodulating agent (such that the component and/or modulating agent isgenerated in situ) in combination with a physiologically acceptablecarrier. In such pharmaceutical compositions, the polynucleotide may bepresent within any of a variety of delivery systems known to those ofordinary skill in the art, including nucleic acid, bacterial and viralexpression systems, as well as colloidal dispersion systems, includingliposomes. Appropriate nucleic acid expression systems contain thenecessary polynucleotide sequences for expression in the patient (suchas a suitable promoter and terminating signal). DNA may also be “naked,”as described, for example, in Ulmer et al., Science 259:1745-49, 1993.

[0074] Various viral vectors that can be used to introduce a nucleicacid sequence into the targeted patient's cells include, but are notlimited to, vaccinia or other pox virus, herpes virus, retrovirus, oradenovirus. Techniques for incorporating DNA into such vectors are wellknown to those of ordinary skill in the art. Preferably, the retroviralvector is a derivative of a murine or avian retrovirus including, butnot limited to, Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A retroviral vector may additionally transfer orincorporate a gene for a selectable marker (to aid in the identificationor selection of transduced cells) and/or a gene that encodes the ligandfor a receptor on a specific target cell (to render the vector targetspecific). For example, retroviral vectors can be made target specificby inserting a nucleotide sequence encoding a sugar, a glycolipid, or aprotein. Targeting may also be accomplished using an antibody, bymethods known to those of ordinary skill in the art.

[0075] Viral vectors are typically non-pathogenic (defective),replication competent viruses, which require assistance in order toproduce infectious vector particles. This assistance can be provided,for example, by using helper cell lines that contain plasmids thatencode all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR, but that are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsulation. Such helper cell lines include (butare not limited to) Ψ2, PA317 and PA12. A retroviral vector introducedinto such cells can be packaged and vector virion produced. The vectorvirions produced by this method can then be used to infect a tissue cellline, such as NIH 3T3 cells, to produce large quantities of chimericretroviral virions.

[0076] Another targeted delivery system for polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. A preferred colloidal system for use as adelivery vehicle in vitro and in vivo is a liposome (i.e., an artificialmembrane vesicle). It has been shown that large unilamellar vesicles(LUV), which range in size from 0.2-4.0 μm can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand be delivered to cells in a biologically active form (Fraley, et al.,Trends Biochem. Sci. 6:77, 1981). In addition to mammalian cells,liposomes have been used for delivery of polynucleotides in plant, yeastand bacterial cells. In order for a liposome to be an efficient genetransfer vehicle, the following characteristics should be present: (1)encapsulation of the genes of interest at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques 6:882, 1988).

[0077] The targeting of liposomes can be classified based on anatomicaland mechanistic factors. Anatomical classification is based on the levelof selectivity and may be, for example, organ-specific, cell-specific,and/or organelle-specific. Mechanistic targeting can be distinguishedbased upon whether it is passive or active. Passive targeting utilizesthe natural tendency of liposomes to distribute to cells of thereticuloendothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0078] Routes and frequency of administration, as well doses, will varyfrom patient to patient. In general, the pharmaceutical compositions maybe administered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity or transdermally. Between 1 and 6 doses maybe administered daily. A suitable dose is an amount that is sufficientto show improvement in the symptoms of a patient afflicted with adisease associated with the NF-κB cascade. Such improvement may bedetected by monitoring inflammatory responses (e.g., edema, transplantrejection, hypersensitivity) or through an improvement in clinicalsymptoms associated with the disease. The dosage may generally varydepending on the nature of the modulating agent and the disease to betreated. Typically, the amount of modulating agent present in a dose, orproduced in situ by DNA present in a dose, ranges from about 1 μg toabout 200 mg per kg of host. Suitable dose sizes will vary with the sizeof the patient, but will typically range from about 10 mL to about 500mL for 10-60 kg animal.

[0079] In another aspect, the present invention provides methods fordetecting the level of stimulus-inducible IκB kinase activity in asample. The level of IκB kinase activity may generally be determined viaan immunokinase assay, in which IKK signalsome is firstimmunoprecipitated with an antibody that binds to the complex. Theimmunoprecipitated material is then subjected to a kinase assay asdescribed herein. Substrate specificity may be further evaluated asdescribed herein to distinguish the activity of a stimulus-inducible IκBkinase complex from other kinase activities.

[0080] The present invention also provides methods for detecting thelevel of IKK signalsome, or a component thereof, in a sample. The amountof IKK signalsome, IκB kinase or nucleic acid encoding IκB kinase, maygenerally be determined using a reagent that binds to IκB kinase, or toDNA or RNA encoding a component thereof. To detect nucleic acid encodinga component, standard hybridization and/or PCR techniques may beemployed using a nucleic acid probe or a PCR primer. Suitable probes andprimers may be designed by those of ordinary skill in the art based onthe component sequence To detect IKK signalsome or a component thereof,the reagent is typically an antibody, which may be prepared as describedbelow.

[0081] There are a variety of assay formats known to those of ordinaryskill in the art for using an antibody to detect a protein in a sample.See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988. For example, the antibody may be immobilized ona solid support such that it can bind to and remove the protein from thesample. The bound protein may then be detected using a second antibodythat binds to the antibody/protein complex and contains a detectablereporter group. Alternatively, a competitive assay may be utilized, inwhich protein that binds to the immobilized antibody is labeled with areporter group and allowed to bind to the immobilized antibody afterincubation of the antibody with the sample. The extent to whichcomponents of the sample inhibit the binding of the labeled protein tothe antibody is indicative of the level of protein within the sample.Suitable reporter groups for use in these methods include, but are notlimited to, enzymes (e.g., horseradish peroxidase), substrates,cofactors, inhibitors, dyes, radionuclides, luminescent groups,fluorescent groups and biotin.

[0082] Antibodies encompassed by the present invention may be polyclonalor monoclonal, and may bind to IKK signalsome and/or one or morecomponents thereof (e.g., IKK-1 and/or IKK-2). Preferred antibodies arethose antibodies that inhibit or block IκB kinase activity in vivo andwithin an in vitro assay, as described above. Other preferred antibodiesare those that bind to one or more IκB proteins. As noted above,antibodies and other agents having a desired effect on IκB kinaseactivity may be administered to a patient (either prophylactically orfor treatment of an existing disease) to modulate the phosphorylation ofan IκB, such as IκBα, in vivo.

[0083] Antibodies may be prepared by any of a variety of techniquesknown to those of ordinary skill in the art (see, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).In one such technique, an immunogen comprising the protein of interestis initially injected into a suitable animal (e.g., mice, rats, rabbits,sheep and goats), preferably according to a predetermined scheduleincorporating one or more booster immunizations, and the animals arebled periodically. Polyclonal antibodies specific for the protein maythen be purified from such antisera by, for example, affinitychromatography using the protein coupled to a suitable solid support.

[0084] Monoclonal antibodies specific for an IKK signalsome or acomponent thereof may be prepared, for example, using the technique ofKohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvementsthereto. Briefly, these methods involve the preparation of immortal celllines capable of producing antibodies having the desired specificity(i.e., reactivity with the complex and/or component of interest). Suchcell lines may be produced, for example, from spleen cells obtained froman animal immunized as described above. The spleen cells are thenimmortalized by, for example, fusion with a myeloma cell fusion partner,preferably one that is syngeneic with the immunized animal. For example,the spleen cells and myeloma cells may be combined with a nonionicdetergent for a few minutes and then plated at low density on aselective medium that supports the growth of hybrid cells, but notmyeloma cells. A preferred selection technique uses HAT (hypoxanthine,aminopterin, thymidine) selection. After a sufficient time, usuallyabout 1 to 2 weeks, colonies of hybrids are observed. Single coloniesare selected and tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

[0085] Monoclonal antibodies may be isolated from the supernatants ofgrowing hybridoma colonies. In addition, various techniques may beemployed to enhance the yield, such as injection of the hybridoma cellline into the peritoneal cavity of a suitable vertebrate host, such as amouse. Monoclonal antibodies may then be harvested from the ascitesfluid or the blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction.

[0086] In a related aspect of the present invention, kits for detectingthe level of IKK signalsome, IκB kinase, nucleic acid encoding IκBkinase and/or IκB kinase activity in a sample are provided. Any of avariety of samples may be used in such assays, including eukaryoticcells, bacteria, viruses, extracts prepared from such organisms andfluids found within living organisms. In general, the kits of thepresent invention comprise one or more containers enclosing elements,such as reagents or buffers, to be used in the assay.

[0087] A kit for detecting the level of IKK signalsome, IκB kinase ornucleic acid encoding IκB kinase typically contains a reagent that bindsto the compound of interest. To detect nucleic acid encoding IκB kinase,the reagent may be a nucleic acid probe or a PCR primer. To detect IKKsignalsome or IκB kinase, the reagent is typically an antibody. Suchkits also contain a reporter group suitable for direct or indirectdetection of the reagent (i.e., the reporter group may be covalentlybound to the reagent or may be bound to a second molecule, such asProtein A, Protein G, immunoglobulin or lectin, which is itself capableof binding to the reagent). Suitable reporter groups include, but arenot limited to, enzymes (e.g., horseradish peroxidase), substrates,cofactors, inhibitors, dyes, radionuclides, luminescent groups,fluorescent groups and biotin. Such reporter groups may be used todirectly or indirectly detect binding of the reagent to a samplecomponent using standard methods known to those of ordinary skill in theart.

[0088] In yet another aspect, IKK signalsome may be used to identify oneor more native upstream kinases (i.e., kinases that phosphorylate andactivate IKK signalsome in vivo) or other regulatory molecules thataffect IκB kinase activity (such as phosphatases or molecules involvedin ubiquitination), using methods well known to those of ordinary skillin the art. For example, IKK signalsome components may be used in ayeast two-hybrid system to identify proteins that interact with suchcomponents. Alternatively, an expression library may be screened forcDNAs that phosphorylate IKK signalsome or a component thereof.

[0089] The following Examples are offered by way of illustration and notby way of limitation.

EXAMPLES Example 1 Recruitment of NFκB into IKK Sigmalsome DuringActivation

[0090] This example illustrates the recruitment of NFκB into a proteincomplex (the IKK signalsome) containing IκB kinase and other signalingproteins.

[0091] Cytoplasmic extracts of either unstimulated or stimulated Jurkatcells were fractionated on a Superdex 200 gel filtration column, andIκBα was visualized by immunoblot analysis. Jurkat cells were grown to acell density of 1.5×10⁶ cells/ml and either not stimulated or inducedfor 10 minutes with PMA (50 ng/ml)/PHA (1 μg/ml). Cells were harvestedand resuspended in two volumes HLB buffer (20 mM Tris pH 8.0, 2 mM EDTA,1 mM EGTA, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μMNa₃VO₄, 1 mM benzamidine, 2 mM PMSF, 10 μg/ml aprotonin, 1 μg/mlleupeptin, 1 μg/ml pepstatin, 1 mM DTT), made 0.1% NP40 and left on icefor 15 minutes, and lysed with a glass Dounce homogenizer. The nucleiwere pelleted at 10,000 rpm for 20 minutes in a Sorval SS34 rotor. Thesupernatant was further centrifuged at 40,000 rpm for 60 min in a Ti50.1rotor. All procedures were carried out at 4° C. The S-100 fraction wasconcentrated and chromatographed on Hi Load 16/60 Superdex 200 prepgrade gel filtration column that was equilibrated in GF buffer (20 mMTris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5% glycerol, 0.025%Brij 35, 1 mM benzamidine, 2 mM PMSF, 10 mM β-glycerophosphate, 10 mMNaF, 10 mM PNPP, 300 μM Na₃VO₄, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1μg/ml pepstatin, 1 mM DTT). Isolated fractions were analyzed by westernblot analysis using either anti-IκBα or anti-JNK antibodies (Santa Cruz,Inc., Santa Cruz, Calif.).

[0092] As shown in FIG. 1A, IκBα in cell extracts from unstimulatedcells eluted with an apparent molecular weight of ˜300 kDa (lanes 5-7),consistent with the chromatographic properties of the inactive NFκB-IκBcomplex (Baeuerle and Baltimore, Genes Dev. 3:1689-98, 1989). Incontrast, phosphorylated IκBα (from cells stimulated for periods tooshort to permit complete degradation of the protein) migrated at ˜600kDa on the same chromatography columns (lanes 2, 3). This difference inmigration was specific for IκB, since analysis of the same fractionsindicated that the Jun N-terminal kinases JNK1 and JNK2 migrated withlow apparent molecular weight and showed no difference inchromatographic behavior between stimulated and unstimulated cells.Stimulation-dependent recruitment of IκB into this larger proteincomplex corresponded with the appearance of phosphorylated IκB,suggesting that the complex contained the specific IκB kinases thatmediate IκB phosphorylation. These results demonstrate that that NFκBactivation involves recruitment into a protein complex (the IKKsignalsome) containing IκB kinase and other signaling proteins.

Example 2 Partial Purification of IKK Signalsome and Identification ofCo-Purifying Components

[0093] This Example illustrates the fractionation of extracts containingIκB kinase. Whole cell extracts from TNFα-stimulated cells werefractionated by gel filtration, ion exchange, and other chromatographicmethods, as described above. IκB kinase activity in the fractions wasassayed by phosphorylation of GST-IκBα (1-54) (SEQ ID NO:3) or GST-IκB β(1-44) (SEQ ID NO:4). Kinase assays were performed in 20 mM HEPES pH7.7, 2 MM MgCl₂, 2 mM MnCl₂, 10 μM ATP, 1-3 μCi γ-[³²P]-ATP, 10 mMβ-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na₃VO₄, 1 mMbenzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1 μg/mlpepstatin, 1 mM DTT) at 30° C. for 30 to 60 minutes in the presence ofthe indicated substrate. The kinase reaction was stopped by the additionof 6×SDS-PAGE sample buffer, subjected to SDS-PAGE analysis andvisualized using autoradiography. GST-IκB substrates for use in theabove assay were prepared using standard techniques for bacteriallyexpressed GST-protein (see Current Protocols in Molecular Biology2:16.7.1-16.7.7, 1996). Bacterial cells were lysed, GST proteins werepurified via binding to GST-agarose beads, washed several times, elutedfrom the beads with glutathione, dialyzed against kinase assay bufferand stored at −80° C. The specificity of the kinase was established byusing mutant GST-IκBα (1-54) in which serines 32, 36 had been mutated tothreonine (SEQ ID NO:5), and GST-IκBβ (1-44) in which serines 19, 23 hadbeen mutated to alanine (SEQ ID NO:6).

[0094] IκB kinase activity was not observed in extracts fromunstimulated cells, while stimulation with TNFα for 5-7 minutes resultedin strong induction of kinase activity. As shown in FIG. 1B, the IκBkinase activity from stimulated cells chromatographed on gel filtrationas a broad peak of ˜500-700 kDa, consistent with its presence in a largeprotein complex potentially containing other components required forNFκB activation.

[0095] NFκB activation is known to occur under conditions that alsostimulate MAP kinase pathways (Lee et al., Cell 88:213-22, 1997; Hirano,et al., J. Biol. Chem. 271:13234-38, 1996). Accordingly, furtherexperiments were performed to detect proteins associated with MAP kinaseand phosphatase cascades at various stages of purification of the IKKsignalsome. In addition to RelA and IκBβ, MEKK-1 and twotyrosine-phosphorylated proteins of ˜55 and ˜40 kDa copurified with IκBkinase activity (FIG. 1C). Antibodies to Rel A and IκBβ were obtainedfrom Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.), and antibodiesto MEKK-1 were obtained from Upstate Biotechnology (Lake Placid, N.Y.).Other signaling components, including PKCζ, PP1 and PP2A, were detectedin the same fractions as the IκB kinase in early chromatographic stepsbut did not copurify at later chromatographic steps (data not shown).Most interestingly, an unidentified protein of ˜50 kDa, detected by itscrossreaction with an antibody to MKP-1, copurified with IκB kinasethrough several purification steps (FIG. 1C). This protein is unlikelyto be MKP-1 itself, since the molecular weight of authentic MKP-1 is 38kDa.

Example 3 Preparation of IKK Signalsome from HeLa S3 Cell Extracts

[0096] This Example illustrates an alternate preparation of an IKKsignalsome, and the characterization of the complex.

[0097] HeLa S3 cells were grown to a cell density of approximately0.6×10⁶/mL, concentrated 10 fold and induced with TNFα (30 ng/mL) forseven minutes. Two volumes of ice-cold PBS containing phosphataseinhibitors (10 mM sodium fluoride, 0.3 mM sodium orthovanadate and 20 mMβ-glycerophosphate) were then added. The cells were spun down, washedonce with ice-cold PBS containing phosphatase inhibitors and snapfrozen.

[0098] Standard protocols were then used to prepare cytoplasmic andnuclear extracts. More specifically, the frozen HeLa S3 cell pellet wasquick-thawed at 37° C., resuspended in 2 volumes of ice-cold HypotonicLysis Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 1 mM EGTA, 10 mMβ-glycerophosphate, 10 mM NaF, 10 mM PNPP, 0.3 mM Na₂VO₄, 5 mM sodiumpyrophosphate, 1 mM benzamidine, 2 mM PMSF, 10 μg/mL aprotinin, 1 μg/mLleupeptin and 1 μg/mL pepstatin), and left to incubate on ice for 30min. The swollen cells were then dounced 30 times using a tight pestleand the nuclei were pelleted at 10,000 rpm for 15 minutes at 4° C. Thesupernatant was clarified via ultracentrifugation (50,000 rpm for 1 hourat 4° C.) to generate the final cytoplasmic extract. Thenuclear/membrane pellet was resuspended in an equal volume of High SaltExtraction Buffer (20 mM Tris pH 8.0, 0.5M NaCl, 1 mM EDTA, 1 mM EGTA,0.25% Triton X-100, 20 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 0.3mM Na₂VO₄, 1 mM benzamidine, 1 mM PMSF, 1 mM DTT, 10 μg/mL aprotinin, 1μg/mL leupeptin and 1 μg/mL pepstatin) and allowed to rotate at 4° C.for 30 minutes. Cell debris was removed via centrifugation at 12,500 rpmfor 30 minutes at 4° C. and the resulting supernatant was saved as thenuclear/membrane extract.

[0099] These extracts were then independently subjected to a series ofchromatographic steps (shown in FIG. 2) using a Pharmacia FPLC system(Pharmacia Biotech, Piscataway, N.J.):

[0100] (1) Q Sepharose (Pharmacia Biotech, Piscataway, N.J.)—the columnwas run with a linear gradient starting with 0.0M NaCl Q Buffer (20 mMTris pH 8.0, 0.5 mM EDTA, 0.5 mM EGTA, 0.025% Brij 35, 20 mMβ-glycerophosphate, 10 mM NaF, 0.3 mM Na₂VO₄, 1 mM benzamidine, 1 mMPMSF, 2 mM DTT, 10 μg/mL aprotinin, 1 μg/mL leupeptin and 1 μg/mLpepstatin) and ending with 0.5M NaCl Q Buffer. The IκBα kinase activityeluted between 0.25 and 0.4 M NaCl.

[0101] (2) Gel Filtration HiLoad 16/60 Superdex 200) (Pharmacia Biotech,Piscataway, N.J.)—the column was run with Gel Filtration Buffer (20 mMTris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.05% Brij 35, 20 mMβ-glycerophosphate, 10 mM NaF, 0.3 mM Na₂VO₄, 1 mM benzamidine, 1 mMPMSF, 1 mM DTT, 10 μg/mL aprotinin, 1 μg/mL leupeptin and 1 μg/m Lpepstatin). The peak IκBα kinase activity eluted at 40-48 mL, whichcorresponds to a molecular weight of 731 kD to 623 kD.

[0102] (3) HR 5/5 Mono Q (Pharmacia Biotech, Piscataway, N.J.)—thecolumn was run with a linear gradient starting with 0.0M NaCl Q Bufferand ending with 0.5M NaCl Q Buffer (without Brij detergent to preparesample for Phenyl Superose column). The IκBα kinase activity elutedbetween 0.25 and 0.4 M NaCl.

[0103] (4) HR Phenyl Superose (Pharmacia Biotech, Piscataway, N.J.)—thecolumn was run with a linear gradient of 1.0M to 0.0M ammonium sulfatein Phenyl Superose Buffer (20 mM Tris pH 8.0, 0.25 mM EDTA, 1 mM EGTA,20 mM β-glycerophosphate, 10 mM NaF, 0.1 mM Na₂VO₄, 1 mM benzamidine, 1mM PMSF, 1 mM DTT, 10 μg/mL aprotinin, 1 μg/mL leupeptin and 1 μg/mLpepstatin). The IκBα kinase activity eluted between 0.35 and 0.2 Mammonium sulfate.

[0104] (5) Gel Filtration Superdex 200 HR 10/30 (Pharmacia Biotech,Piscataway, N.J.)—the column was run with Gel Filtration Buffer (see(2), above). The peak of activity eluted at 8-10 mL, which correspondsto a molecular weight of 720 kD to 600 kD.

[0105] (6) HR 5/5 Mono Q—the column was run as in (3) above except thatthe 0.05% Brij 35 was included in all Q buffers.

[0106] IκBα kinase activity, with similar substrate specificity andmolecular weight, was isolated from both the cytoplasmic andnuclear/membrane extracts.

[0107] At each step of the fractionation, IκB kinase activity wasmonitored using an in vitro assay. The assay was performed by combining2 μg of the respective IκB substrate (GST-IκBα 1-54 (wildtype) orGST-IκBα (S32/36 to T), as described below) with 3-5 μL chromatographicfraction and 20 μL of Kinase Assay Buffer (20 mM HEPES pH 7.4, 10 mMMgCl₂, 10 mM MnCl₂, 20 mM NaCl, 1 mM DTT, 20 mM PNPP, 20 μM ATP, 20 mMα-glycerophosphate, 10 mM NaF, 0.1 mM Na₂VO₄, 1 mM benzamidine, 1 mMPMSF) containing γ³²P-ATP, and incubating for 30 minutes at 30° C. Thekinase reaction was terminated by adding 8 μL of 6×SDS-PAGE samplebuffer. The entire sample was run on a 12% polyacrylamide gel, dried andsubjected to autoradiography.

[0108] IκB substrates for use in the above assay were prepared usingstandard techniques (see Haskill et al., Cell 65:1281-1289, 1991). TheGST-IκBα 1-54 (wildtype) or GST-IκBα (S32/36 to T) substrates wereprepared using standard techniques for bacterially expressedGST-protein. Bacterial cells were lysed, GST proteins were purified viabinding to GST-agarose beads, washed several times, eluted from thebeads with glutathione, dialyzed against 50 mM NaCl Kinase Assay Bufferand stored at −80° C.

[0109] The TNFα-inducibility of IκB kinase activity was initiallyevaluated by Western blot analysis of the levels of IκB in HeLa S3cytoplasmic extracts following gel filtration. IκBα was assayed byrunning 18 μL of the gel filtration fractions on 10% SDS PAGE,transferring to Nitrocellulose Membrane (Hybond-ECL, Amersham LifeSciences, Arlington Height, Ill.) using standard blotting techniques andprobing with anti-IκBα antibodies (Santa Cruz Biotechnology, Inc., SantaCruz, Calif.). TNFα-inducibility was evaluated by comparing the level ofIκBα in cells that were (FIG. 3B) and were not (FIG. 3A) exposed to TNFα(30 ng/mL for seven minutes, as described above).

[0110] The IκB kinase activity of these cytoplasmic extracts wasevaluated using the kinase assay described above. As shown in FIG. 4B,the extract of TNFα-treated cells phosphorylated GST-IκBα 1-54(wildtype), while the untreated cell extract showed significantly lowerlevels of IκBα kinase activity (FIG. 4A).

[0111] Cytoplasmic extracts of TNFα-treated HeLa S3 cells (following QSepharose fractionation) were also subjected to in vitro kinase assays,using the N-terminal portion of IκBα (residues 1 to 54) as substrate.With the wild type substrate, phosphorylation of GST-IκBα 1-54 wasreadily apparent (FIG. 5A). In contrast, substrate containing threoninesubstitutions at positions 32 and 36 was not phosphorylated (FIG. 5B).

[0112] Following chromatographic fractionation by Q Sepharose, Superdex200, MonoQ Sepharose and Phenyl Superose, in vitro kinase assay showedsubstantial purification of the IκB kinase activity (FIG. 6A). Furtherpurification of the IκB kinase was achieved by passing the sample over,in series, an analytical Superdex 200 and Mono Q HR 5/5, resulting in 8major protein bands as determined by silver staining. As before, the useof substrate containing threonine substitutions at positions 32 and 36markedly reduced the phosphorylation (FIG. 6B). These resultsdemonstrate the purification of a stimulus-inducible IκBα kinasecomplex, which specifically phosphorylates serine residues at positions32 and 36 of IκBα without the addition of exogenous factors.

Example 4 Immunoprecipitation of IKK Signalsome Using Anti MKP-1Antibodies

[0113] This Example illustrates the immunoprecipitation of IκB kinaseactivity from cytoplasmic extracts prepared from stimulated cells.

[0114] A. Immunoprecipitation of IκB Kinase Complex from HeLa Cells

[0115] HeLa cells were TNF-α-treated (30 μg/mL, 7 minutes) andfractionated by gel filtration as described in Example 3. Twenty μL ofgel filtration fraction #6 (corresponding to about 700 kD molecularweight) and 1 μg purified antibodies raised against MKP-1 (Santa CruzBiotechnology, Inc., Santa Cruz, Calif.) were added to 400 μL of icecold 1×Pull Down Buffer (20 mM Tris pH 8.0,250 mM NaCl, 0.05% NP-40, 3mM EGTA, 5 mM EDTA, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300μM Na₃VO₄, 1 mM benzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/mlleupeptin, 1 μg/ml pepstatin, 1 mM DTT). The sample was gently rotatedfor 1 hour at 4° C., at which time 40 μL of protein A-agarose beads(50:50 slurry, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) wasadded. The sample was then rotated for an additional 1.5 hours at 4° C.The protein A-agarose beads were pelleted at 3,000 rpm for 2 minutes at4° C. and the pellet was washed three times with ice cold Pull DownBuffer (800 μL per wash).

[0116] The pellet was subjected to the standard in vitro IκBα kinaseassay (as described above) using either 2 μg GST-IκBα1-54 (wildtype) or2 μg-GST-IκBα1-54 (S32/36 to T) as the substrate.

[0117] The results, shown in FIG. 7, demonstrate that antibodiesdirected against MKP-1 immunoprecipitate the stimulus-inducible IκBαkinase activity. The substrate specificity of this IκKα kinase activitycorresponds to what has been described in vivo (strong phosphorylationof the GST-IκBα 1-54 (wildtype) and no phosphorylation usingGST-IκBα1-54 (S32/36 to T).

[0118] B. Characterization of Immunoprecipitated IKK Signalsome

[0119] For these studies, small scale immunoprecipitation were performedusing two 150 mm plates of HeLa cells (one stimulated and oneunstimulated). Whole cell lysates were diluted 4-fold with 2×Pull-DownBuffer (40 mM Tris pH 8.0, 500 mM NaCl, 0.1% NP-40, 6 mM EDTA, 6 mMEGTA, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na₃VO₄, 1mM benzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1μg/ml pepstatin, 1 mM DTT) and 2-4 μg of the indicated antibody wasadded. Lysates were incubated on ice for 1-2 hours, 10 μl of Protein Aor G beads were added, and lysates were left to incubate with gentlerotation for an additional 1 hour at 4° C. The immunoprecipitate wasthen washed 3 times with 2×Pull-Down Buffer, 1×with kinase bufferwithout ATP and subjected to a kinase assay as described in Example 2.There was no noticeable loss in IκB kinase activity when theimmunoprecipitate was subjected to more rigorous washing, such as inRIPA buffer (20 mM Tris, 250 mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS, 3 mMEDTA, 3 mM EGTA. 10 mM, β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300μM Na₃VO₄, 1 mM benzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/mlleupeptin, 1 μg/ml pepstatin, 1 mM DTT) or washes up to 3.5 M urea.

[0120] Of a large panel of antibodies tested, one of three anti-MKP-1antibodies efficiently co-immunoprecipitated an inducible IκB kinaseactivity from HeLa cells as well as primary human umbilical veinendothelial cells (HUVEC). The co-immunoprecipitated kinase (IKKsignalsome kinase) was inactive in unstimulated HeLa cells, but wasrapidly activated within minutes of TNFα stimulation (FIG. 8A, toppanel). The IKK signalsome kinase did not phosphorylate a mutantGST-IκBα protein in which serine residues 32 and 36 had been mutated tothreonine (FIG. 8A top panel, even-numbered lanes). Activation of thesignalsome kinase was maximal at 5 minutes and declined thereafter, atime course consistent with the time course of IκBα phosphorylation anddegradation under the same conditions (FIG. 8A, bottom panel). Asexpected, the signalsome IκB kinase was also activated by stimulation ofcells with IL-1 or PMA (FIG. 8B, lanes 1-4); moreover, its activity wasinhibited in cells treated with TPCK, a known inhibitor of NFκBactivation (FIG. 8B, lane 7). Additionally, the IKK signalsome kinasespecifically phosphorylated full-length wild-type IκBα, but not a mutantIκBα bearing the serine 32, 36 to alanine mutations, in the context of aphysiological RelA-IκBα complex (FIG. 8C, lanes 3, 4). Together theseresults indicate that the anti-MKP-1 antibody co-immunoprecipitated theIKK signalsome. The signalsome-associated IκB kinase met all thecriteria expected of the authentic IκB kinase and had no detectable IκBαC-terminal kinase activity.

[0121] The specificity of the IKK signalsome kinase was furtherestablished by kinetic analysis and by examining its activity on variouspeptides and recombinant protein substrates (FIG. 9A). For thesestudies, synthetic peptides (Alpha Diagnostics International, SanAntonio, Tex.) were prepared with the following sequences:

[0122] IκBα (21-41): CKKERLLDDRHDSGLDSMKDEE (SEQ ID NO:11)

[0123] IκBα (21-41) S/T mutant: CKKERLLDDRHDTGLDTMKDEE (SEQ ID NO:12)

[0124] c-Fos(222-241): DLTGGPEVAT(PO3)PESEEAFLP (SEQ ID NO:13)

[0125] MKP-1: CPTNSALNYLKSPITTSPS (SEQ ID NO:14)

[0126] cJun (56-70): CNSDLLTSPDVGLLK (SEQ ID NO:15)

[0127] cJun (65-79): CVGLLKLASPELERL (SEQ ID NO:16)

[0128] Phosphorylation of these peptides (100 μM) was performed using akinase reaction as described above. Reactions were for one hour at roomtemperature and were terminated by the addition of SDS-PAGE loadingbuffer. SDS-PAGE with a 16% Tris/tricine gel (Novex, San Diego, Calif.)or a 4-20% Tris/glycine gel (Novex, San Diego, Calif.) was used tocharacterize the reaction products. Gels were washed, dried in vacuo,and exposed to autoradiographic film.

[0129] Inhibition of immunopurified IKK signalsome activity was measuredby ³²P incorporation into GST-IκKα (1-54) in a discontinuous assay usingthe reaction conditions described above. The concentrations of GST-IκBα(1-54) and ATP used in the inhibition studies were 0.56 μM and 3 μM,respectively. Enzymatic reactions (32 μL) were carried out in wells of a96 well assay plate for one hour at room temperature and terminated withthe addition of trichloroacetic acid (TCA) (150 μL/well of 12.5% w/v).The subsequent 20 minute incubation with TCA precipitated the proteinsbut not peptides from solution. The TCA precipitate was collected on96-well glass fiber plates (Packard) and washed 10 times withapproximately 0.3 mL per well of Dulbecco's phosphate buffered saline pH7.4 (Sigma) using a Packard Filtermate 196. Scintillation fluid (0.50mL, MicroScint, Packard) was added to each well and the plate wasanalyzed for ³²P using a Packard TopCount scintillation counter. Lessthan 10% of ATP was turned over in the course of the assay reaction,ensuring that the kinetic data represented initial rate data. Theinhibition constant of the P32, 36 peptide was determined by Dixonanalysis (Dixon and Webb, In Enzymes (Academic Press: New York, ed. 3,1979), pp. 350-51.

[0130] The kinase displayed normal Michaelis-Menten kinetics, suggestingthat it was not a mixture of diverse unrelated kinases. The kinase wascapable of phosphorylating an IκBα (21-41) peptide (FIGS. 9A and 9B)) aswell as two different IκBα (21-41) peptides, each bearing a free serineat either position 32 or 36 and phosphoserine at the other position(FIGS. 9A and 9B, lanes 2, 3). As expected, a peptide withphosphoserines at both positions was not phosphorylated at all (FIG. 9B,top), indicating that there was no significant turnover of thephosphates under our reaction conditions. These experiments indicatedthat both serines 32 and 36 were phosphoacceptor sites for the IKKsignalsome kinase, thus distinguishing it from other kinases such aspp90Rsk which phosphorylates IκBα only at serine 32 (Schouten, et al.,EMBO J. 16:3133-44, 1997).

[0131] Although the IKK signalsome kinase efficiently phosphorylated IκBpeptides, it did not phosphorylate the c-Fos phosphopeptide containing afree serine and a free threonine (FIG. 9B, top), two c-Jun peptidescontaining serine 63 and 73, respectively, (FIG. 9A, top panel, lanes 4,5), or an MKP-1 peptide containing four serines and three threonines(FIG. 9A, lane 6). The latter peptides were substrates for JNK2 (FIG.9A, bottom panel, lanes 4-6). An IκBα (21-41) peptide in which serines32 and 36 were replaced by threonines was phosphorylated by thesignalsome at least 10-fold less well than the wild-typeserine-containing peptide, consistent with the slower phosphorylationand degradation kinetics of IκBα (S32/36 to T) in cells (DiDonato etal., Mol Cell. Biol. 16:1295-1304, 1996), and the preference of thekinase for serine over threonine at positions 32, 36 in both full-lengthIκBα and GST-IκBα (1-54) (FIGS. 8A and C). In addition, the kinasephosphorylated GST-IκBβ (1-54), albeit with lower affinity. In mostexperiments, IκB kinase activity was also associated with strong RelAkinase activity (FIG. 8C, lanes 3, 4), but no activity was observedtowards several other substrates including myelin basic protein (MBP),GST-ATF2 (1-112), GST-cJun (1-79), GST-ERK3, GST-Elk (307428), GST-p38,and a GST fusion protein containing the C-terminal region of IκBα(242-314).

[0132] The specificity of the IKK signalsome kinase was furtheremphasized by its susceptibility to product inhibition (FIG. 9B,bottom). The kinase was strongly inhibited by a doubly-phosphorylatedIκBα peptide bearing phosphoserines at both positions 32 and 36, but notby the unrelated c-Fos phosphopeptide that contained a singlephosphothreonine. The singly-phosphorylated and the unphosphorylatedIκBα peptides were less effective inhibitors.

Example 5 Absence of Free Ubiguitin in Purified IKK Signalsome

[0133] This example illustrates the absence of detectable free ubiquitinwith a IKK signalsome prepared as in Example 3. Standard western blotprocedures were performed (Amersham Life Science protocol, ArlingtonHeights, Ill.). 100 ng ubiquitin, 10 ng ubiquitin and 20 ul purifiedIκBα kinase complex was subjected to 16% Tricine SDS-PAGE (Novex, SanDiego, Calif.), transferred to Hybond ECL Nitrocellulose membrane(Amersham Life Science, Arlington Heights, Ill.), and probed withantibodies directed against ubiquitin (MAB1510; Chemicon, Temecula,Calif.). The results are shown in FIG. 10. Free ubiquitin could not bedetected in the purified IκBα kinase preparation (even at very longexposures). The complexes described herein do not require addition ofendogenous ubiquitin to detect IκBα kinase activity, nor is freeubiquitin a component in the purified IκBα kinase preparations of thepresent invention.

Example 6 Purification of the NFκB Signalsome and Identification ofIKK-1 and IKK-2

[0134] This Example illustrates a two-step affinity method forpurification of the IKK signalsome, based on its recognition by theMKP-1 antibody (depicted in FIG. 11A) and the identification of IκBkinases.

[0135] For large scale IKK signalsome purification, HeLa S3 cells werestimulated for 7 minutes with 20 ng/ml TNFα (R&D Systems, Minneapolis,Minn.), harvested, whole cell lysates were prepared (1.2 g totalprotein) and approximately 5 mg of anti-MKP-1 antibody (Santa CruzBiotechnology, Inc., Santa Cruz, Calif.) was added and incubated at 4°C. for 2 hours with gentle rotation. Subsequently, 50 ml of Protein Aagarose (Calbiochem, San Diego, Calif.) was added and the mixture wasincubated for an additional 2 hours. The immunoprecipitate was thensequentially washed 4×Pull-Down Buffer, 2×RIPA buffer, 2×Pull-DownBuffer, 1×3.5 M urea-Pull-Down Buffer and 3×Pull-Down Buffer. Theimmunoprecipitate was then made into a thick slurry by the addition of10 ml of Pull-Down Buffer, 25 mg of the specific MKP-1 peptide to whichthe antibody was generated (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.) was added, and the mixture was incubated overnight at 4° C. withgentle rotation. The eluted IKK signalsome was then desalted on PD 10desalting columns (Pharmacia Biotech, Piscataway, N.J.) equilibratedwith 50 mM Q buffer and chromatographed on a Mono Q column (PharmaciaBiotech, Piscataway, N.J.). Fractions containing peak IκB kinaseactivity were pooled, concentrated and subjected to preparativeSDS-PAGE. The intensity of two prominent protein bands of ˜85 and ˜87kDa (indicated by silver stain in FIG. 11B as IKK-1 and IKK-2respectively) correlated with the profile of IκB kinase activity.

[0136] Coomassie stained ˜85 and ˜87 kDa bands were excised, in-geldigested with trypsin (Wilm et al., Nature 37:466-69, 1996) and a smallaliquot of the supernatant was analyzed by high mass accuracy MALDIpeptide mass mapping, as described by Shevchenko et al., Proc. Natl.Acad. Sci. USA 93:14440-45, 1996. The peptide mass map from the IKK-1band was searched against a comprehensive protein sequence databaseusing the program PeptideSearch developed in house at EMBL Heidelberg.Eight measured peptide masses matched calculated tryptic peptide massesfrom CHUK (conserved helix-loop-helix ubiquitous kinase; Connely andMarcu, Cell. Mol. Biol. Res. 41:537-49, 1995) within 30 ppm,unambiguously identifying the protein. The peptide mass map of the IKK-2band did not result in a clear identification and therefore the samplewas subjected to nanoelectrospray mass spectrometry (Wilm and Mann, AnalChem. 68:1-8, 1996). The peptide mixture obtained after extraction ofthe gel piece was micropurified on a capillary containing 50 nL of POROSR2 resin (PerSeptive Biosystems, Framingham, Mass.). After washing, thepeptides were step-eluted with 0.5 μl of 50% MeOH in 5% formic acid intoa nanoelectrospray needle. This needle was transferred to an APIII massspectrometer (Perkin-Elmer, Sciex, Toronto, Canada) and the samplesprayed for approximately 20 minutes. During this time, peptide ionsapparent from the mass spectrum were selected and isolated in turn andfragmented in the collision chamber of the mass spectrometer. From thetandem mass spectra, short stretches of sequence were assembled intopeptide sequence tags (Mann and Wilm, Anal. Chem. 66:4390-99, 1994) andsearched against a protein sequence database or against dbEST usingPeptideSearch.

[0137] Three peptides matched the IKK-1 sequence. A1: IIDLGYAK (SEQ IDNO:17); A2: VEVALSNIK (SEQ ID NO:18); A3 SIQLDLER (SEQ ID NO:19). Threeother peptides matched human EST sequences in dbEST: B1: ALELLPK (SEQ IDNO:20), B2: VIYTQLSK (SEQ ID NO:21), B6: LLLQAIQSFEK (SEQ ID NO:22) allmatch EST clone AA326115. The peptide B4 with the sequence LGTGGFGNVIR(SEQ ID NO:23) was found in clone R06591. After the full-length IKK-2sequence was obtained (as described below) two more peptides B3:ALDDILNLK (SEQ ID NO:24) and B5: DLKPENIVLQQGEQR (SEQ ID NO:25) werefound in the sequence. Peptide A1 is present in both IKK-1 and IKK-2sequences. All the EST clones identified were clearly homologous tohuman and mouse CHUK, a serine/threonine kinase of hitherto unknownfunction. Once the complete coding sequence of IKK-2 was obtained (asdescribed below), all sequenced peptides (apart from two peptidesderived from IKK-1) could be assigned to this protein.

[0138] Representative mass spectra are shown in FIGS. 12A and 12B. InFIG. 12A, peaks labeled A were matched to the tryptic peptides of IKK-1upon fragmentation followed by database searching with peptide sequencetags. Peaks labeled B2, B4, B6 were not found in protein databases butinstead matched human EST sequences. One more peptide (B1) matching ahuman EST clone was observed at m/z 392.2 and is not shown in panel A.In FIG. 12B, a continuous series of C-terminal-containing fragments(Y″-ions) was used to construct a peptide sequence tag as shown by boxedletters. Search of this tag resulted in a match to the peptideLLLQALQSFEK (SEQ ID NO:22) in human EST clone AA326115. Two morepeptides, B1 (ALELLPK; SEQ ID NO:20) and B2 (VIYTQLSK; SEQ ID NO:21)were found in the sequence of the same EST clone.

[0139] Full-length human IKK-1 and IKK-2 cDNAs were cloned based on the.human EST clones, which were obtained from Genome Systems, Inc. (St.Louis, Mo.). The precise nucleotide sequences were determined and usedto design primers to PCR clone human IKK-2 from a human HeLa cell cDNAlibrary (Clontech, Inc., Palo Alto, Calif.). Several IKK-2 cDNA cloneswere isolated and sequenced. Full-length mouse IKK-1 and a partial humanIKK-1 nucleotide sequence was available in the comprehensive database,primers were designed to PCR clone the respective human and mouse IKK-1cDNAs. The partial human IKK-1 coding region was used to probe a HeLacDNA phage library (Stratagene, Inc., La Jolla, Calif.) to obtain thefull-length human IKK-1 cDNA clone using standard procedures.

[0140] Sequence analysis of these clones revealed that IKK-1 and IKK-2were related protein serine kinases (51% identity) containing proteininteraction motifs (FIG. 13A). Both IKK-1 and IKK-2 contain the kinasedomain at the N-terminus, and a leucine zipper motif and ahelix-loop-helix motif in their C-terminal regions (FIG. 13A). Northernanalysis indicated that mRNAs encoding IKK-2 were widely distributed inhuman tissues, with transcript sizes of ˜4.5 kb and 6 kb (FIG. 13B). Thedistribution of IKK-1 (CHUK) transcripts has been reported previously(Connely et al., Cell. Mol. Biol. Res. 41:537-49, 1995). IKK-1 and IKK-2mRNAs are constitutively expressed in Jurkat, HeLa and HUVEC cell lines,and their levels are not altered for up to 8 hours following stimulationwith NFκB inducers such as TNFα (HeLa, HTVEC) or anti-CD28 plus PMA(Jurkat).

[0141] To further characterize the properties of IKK-1 and IKK-2,recombinant HA-tagged IKK-1 and Flag-tagged IKK-2, either separately oralone, were in vitro transcribed and translated in wheat germ or rabbitreticulocyte lysate (Promega, Madison, Wis.). The reactions wereperformed exactly as described in the manufacturer's protocol.Epitope-tagged IKK-1 and IKK-2 then immunoprecipitated with theappropriate anti-tag antibody. Immunoprecipitates containing theseproteins phosphorylated IκBα and IκBβ with the correct substratespecificity (i.e., immunoprecipitates of IKK-1 and IKK-2 phosphorylatedboth GST-IκBα (FIG. 14A, panel 3) and GST-IκBβ (panel 4), but did notphosphorylate the corresponding S32/36 to T mutant protein). IKK-1expressed in rabbit reticulocyte lysates was also capable ofautophosphorylation (FIG. 14A, panel 2, lane 1), whereas akinase-inactive version of IKK-1, in which the conserved lysine 44 hadbeen mutated to methionine, showed no autophosphorylation. In contrastIKK-2, although expressed at equivalent levels in the lysates (panel 1),showed very weak autophosphorylation (panel 2, lane 2).

[0142] Expression of the kinase inactive mutants (K to M) of IKK-1 andIKK-2 indicate that both play critical roles in NFκB activation asdemonstrated by immunofluorescent studies (FIGS. 14B and 14C). For thesestudies, HeLa cells were transiently transfected with either HA-taggedIKK-1 or Flag-tagged IKK-2. Cells were fixed for 30 minutes withmethanol. For immunofluorescence staining, the cells were incubatedsequentially with primary antibody in PBS containing 10% donkey serumand 0.25% Triton X-100 for 2 hours followed by fluorescein-conjugated orTexas red-conjugated secondary antibody (Jackson ImmunoresearchLaboratories, Inc., West Grove, Pa.; used at 1:500 dilution) for 1 hourat room temperature. The coverslips were rinsed and coverslipped withVectashield (Vector Laboratories, Burlingame, Calif.) before scoring andphotographing representative fields. Primary antibodies used forimmunofluorescence staining included antibodies against Rel A (SantaCruz Biotechnology, Inc., Santa Cruz, Calif.), HA tag (Babco, Berkeley,Calif.) and Flag tag (IBI-Kodak, New Haven, Conn.).

[0143] Kinase-inactive versions (K44 to M) of IKK-1 and IKK-2 had noeffect on the subcellular localization of RelA in unstimulated HeLacells, since RelA remained cytoplasmic both in cells expressing theepitope-tagged proteins and in the adjacent untransfected cells (FIGS.14B and 14C, top panels). In contrast, both mutant proteins inhibitedRelA nuclear translocation in TNFα-stimulated cells (FIGS. 14B and 14C,bottom panels). The inhibition mediated by the IKK-2 mutant was strikingand complete (FIG. 14C: compare mutant IKK-2-expressing cells withuntransfected cells in the same field), whereas that mediated by themutant IKK-1 protein, expressed at apparently equivalent levels, wassignificant but incomplete (FIG. 14B). This difference in the functionalactivities of the two mutant kinases may point to distinct roles forthese two kinases in NFκB activation.

[0144] The presence of the leucine zipper and helix-loop-helix motif inIKK-1 and IKK-2 suggested that they interacted functionally with otherproteins in the signalsome. An obvious possibility was that the proteinsformed hetero- or homodimers with one another. HA-tagged IKK-1 andFLAG-tagged IKK-2 were translated in rabbit reticulocyte lysates, eitheralone or together, and then immunoprecipitated with antibodies to theappropriate epitope tags. This experiment demonstrated clearly thatIKK-2 was present in IKK-1 immunoprecipitates (FIG. 15A, lane 3) andvice versa (lane 4), suggesting that these proteins either associateddirectly or via adapter proteins/IKK signalsome components present inthe rabbit reticulocyte lysates. In contrast, however, there was noevidence for association of IKK-1 and IKK-2 that had been cotranslatedin wheat germ lysates (FIG. 15B), suggesting that the proteins did notheterodimerize directly. When full-length IKK-1 was translated togetherin wheat germ extracts with a truncated version of IKK-1 that stillpossessed the protein interaction motifs, there was also no evidence ofassociation, suggesting that IKK-1 was also not capable of forminghomodimers under these conditions.

[0145] Both IKK-1 and IKK-2 kinases were active when expressed in wheatgerm extracts, since they were capable of autophosphorylation, but theywere inactive with respect to phosphorylation of IκB substrates. Sinceboth autophosphorylation and substrate phosphorylation were intact inrabbit reticulocyte lysates, there appeared to be a direct correlationbetween the association of IKK-1 and IKK-2 into a higher order proteincomplex and the presence of specific IκB kinase activity in IKK-1 andIKK-2 immunoprecipitates. This higher order complex is most likely theIKK signalsome itself. Indeed, immunoprecipitation of rabbitreticulocyte lysates with anti-MKP-1 antibody pulls down a low level ofactive IκB kinase activity characteristic of the IKK signalsome.

[0146] It is clear that the IKK signalsome contains multiple proteincomponents in addition to IKK-1 and IKK-2 (FIG. 11B). Some of these maybe upstream kinases such as MEKK-1 (Chen et al., Cell 84:853-62, 1996)or NIK (Malinin, et al., Nature 385:540-44, 1997); others may be adapterproteins that mediate the IKK-1:IKK-2 interaction. Indeed MEKK-1copurifies with IKK signalsome activity (FIG. 1C), and two othersignalsome proteins have been functionally identified. The proteincrossreactive with anti-MKP-1 is an intrinsic component of the IKKsignalsome kinases, since the IκB kinase activity coprecipitated withthis antibody is stable to washes with 2-4 M urea. Moreover, both IKK-1immunoprecipitates and MKP-1 immunoprecipitates containing the IKKsignalsome (FIG. 8C) contain an inducible RelA kinase whose kinetics ofactivation parallel those of the IκB kinase in the sameimmunoprecipitates. Another strong candidate for a protein in thesignalsome complex is the E3 ubiquitin ligase that transfersmultiubiquitin chains to phosphorylated IκB (Hershko et al., Annu. Rev.Biochem. 61:761-807, 1992).

[0147] These results indicate that IKK-1 and IKK-2 are functionalkinases within the IKK signalsome, which mediate IκB phosphorylation andNFκB activation. Appropriate regulation of IKK-1 and IKK-2 may requiretheir assembly into a higher order protein complex, which may be aheterodimer facilitated by adapter proteins, the complete IKKsignalsome, or some intermediate subcomplex that contains both IKK-1 andIKK-2.

[0148] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein for thepurpose of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention.

1 25 317 amino acids amino acid <Unknown> linear 1 Met Phe Gln Ala AlaGlu Arg Pro Gln Glu Trp Ala Met Glu Gly Pro 1 5 10 15 Arg Asp Gly LeuLys Lys Glu Arg Leu Leu Asp Asp Arg His Asp Ser 20 25 30 Gly Leu Asp SerMet Lys Asp Glu Glu Tyr Glu Gln Met Val Lys Glu 35 40 45 Leu Gln Glu IleArg Leu Glu Pro Gln Glu Val Pro Arg Gly Ser Glu 50 55 60 Pro Trp Lys GlnGln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu 65 70 75 80 Ala Ile IleHis Glu Glu Lys Ala Leu Thr Met Glu Val Ile Arg Gln 85 90 95 Val Lys GlyAsp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln 100 105 110 Thr ProLeu His Leu Ala Val Ile Thr Asn Gln Pro Glu Ile Ala Glu 115 120 125 AlaLeu Leu Gly Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly 130 135 140Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val 145 150155 160 Gly Val Leu Thr Gln Ser Cys Thr Thr Pro His Leu His Ser Ile Leu165 170 175 Lys Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala SerIle 180 185 190 His Gly Tyr Leu Gly Ile Val Glu Leu Leu Val Ser Leu GlyAla Asp 195 200 205 Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala LeuHis Leu Ala 210 215 220 Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu LeuLeu Lys Cys Gly 225 230 235 240 Ala Asp Val Asn Arg Val Thr Tyr Gln GlyTyr Ser Pro Tyr Gln Leu 245 250 255 Thr Trp Gly Arg Pro Ser Thr Arg IleGln Gln Gln Leu Gly Gln Leu 260 265 270 Thr Leu Glu Asn Leu Gln Met LeuPro Glu Ser Glu Asp Glu Glu Ser 275 280 285 Tyr Asp Thr Glu Ser Glu PheThr Glu Phe Thr Glu Asp Glu Leu Pro 290 295 300 Tyr Asp Asp Cys Val PheGly Gly Gln Arg Leu Thr Leu 305 310 315 359 amino acids amino acid<Unknown> linear 2 Met Ala Gly Val Ala Cys Leu Gly Lys Thr Ala Asp AlaAsp Glu Trp 1 5 10 15 Cys Asp Ser Gly Leu Gly Ser Leu Gly Pro Asp AlaAla Ala Pro Gly 20 25 30 Gly Pro Gly Leu Gly Ala Glu Leu Gly Pro Glu LeuSer Trp Ala Pro 35 40 45 Leu Val Phe Gly Tyr Val Thr Glu Asp Gly Asp ThrAla Leu His Leu 50 55 60 Ala Val Ile His Gln His Glu Pro Phe Leu Asp PheLeu Leu Gly Phe 65 70 75 80 Ser Ala Gly His Glu Tyr Leu Asp Leu Gln AsnAsp Leu Gly Gln Thr 85 90 95 Ala Leu His Leu Ala Ala Ile Leu Gly Glu AlaSer Thr Val Glu Lys 100 105 110 Leu Tyr Ala Ala Gly Ala Gly Val Leu ValAla Glu Arg Gly Gly His 115 120 125 Thr Ala Leu His Leu Ala Cys Arg ValArg Ala His Thr Cys Ala Cys 130 135 140 Val Leu Leu Gln Pro Arg Pro SerHis Pro Arg Asp Ala Ser Asp Thr 145 150 155 160 Tyr Leu Thr Gln Ser GlnAsp Cys Thr Pro Asp Thr Ser His Ala Pro 165 170 175 Ala Ala Val Asp SerGln Pro Asn Pro Glu Asn Glu Glu Glu Pro Arg 180 185 190 Asp Glu Asp TrpArg Leu Gln Leu Glu Ala Glu Asn Tyr Asp Gly His 195 200 205 Thr Pro LeuHis Val Ala Val Ile His Lys Asp Ala Glu Met Val Arg 210 215 220 Leu LeuArg Asp Ala Gly Ala Asp Leu Asn Lys Pro Glu Pro Thr Cys 225 230 235 240Gly Arg Thr Pro Leu His Leu Ala Val Glu Ala Gln Ala Ala Ser Val 245 250255 Leu Glu Leu Leu Leu Lys Ala Gly Ala Asp Pro Thr Ala Arg Met Tyr 260265 270 Gly Gly Arg Thr Pro Leu Gly Ser Ala Leu Leu Arg Pro Asn Pro Ile275 280 285 Leu Ala Arg Leu Leu Arg Ala His Gly Ala Pro Glu Pro Glu AspGlu 290 295 300 Asp Asp Lys Leu Ser Pro Cys Ser Ser Ser Gly Ser Asp SerAsp Ser 305 310 315 320 Asp Asn Arg Asp Glu Gly Asp Glu Tyr Asp Asp IleVal Val His Ser 325 330 335 Gly Arg Ser Gln Asn Arg Gln Pro Pro Ser ProAla Ser Lys Pro Leu 340 345 350 Pro Asp Asp Pro Asn Pro Ala 355 282amino acids amino acid <Unknown> linear 3 Met Ser Pro Ile Leu Gly TyrTrp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu GluTyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu GlyAsp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro AsnLeu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met AlaIle Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly CysPro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu AspIle Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys Asp Phe GluThr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu LysMet Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly AspHis Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Pro Arg GluPhe 210 215 220 Ile Val Thr Asp Met Phe Gln Ala Ala Glu Arg Pro Gln GluTrp Ala 225 230 235 240 Met Glu Gly Pro Arg Asp Gly Leu Lys Lys Glu ArgLeu Leu Asp Asp 245 250 255 Arg His Asp Ser Gly Leu Asp Ser Met Lys AspGlu Glu Tyr Glu Gln 260 265 270 Met Val Lys Glu Leu Gln Glu Ile Arg Leu275 280 272 amino acids amino acid <Unknown> linear 4 Met Ser Pro IleLeu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg LeuLeu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu ArgAsp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu GluPhe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr GlnSer Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met LeuGly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly AlaVal Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 LysAsp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro LysLeu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile AspLys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln GlyTrp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser AspPro Arg Glu Phe 210 215 220 Ile Val Thr Asp Met Ala Gly Val Ala Cys LeuGly Lys Thr Ala Asp 225 230 235 240 Ala Asp Glu Trp Cys Asp Ser Gly LeuGly Ser Leu Gly Pro Asp Ala 245 250 255 Ala Ala Pro Gly Gly Pro Gly LeuGly Ala Glu Leu Gly Pro Glu Leu 260 265 270 282 amino acids amino acid<Unknown> linear 5 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly LeuVal Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys TyrGlu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn LysLys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile AspGly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile AlaAsp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala GluIle Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val SerArg Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp PheLeu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg LeuCys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro AspPhe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met AspPro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys LysArg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser SerLys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe GlyGly Gly Asp His Pro Pro Lys Ser Asp Pro Arg Glu Phe 210 215 220 Ile ValThr Asp Met Phe Gln Ala Ala Glu Arg Pro Gln Glu Trp Ala 225 230 235 240Met Glu Gly Pro Arg Asp Gly Leu Lys Lys Glu Arg Leu Leu Asp Asp 245 250255 Arg His Asp Thr Gly Leu Asp Thr Met Lys Asp Glu Glu Tyr Glu Gln 260265 270 Met Val Lys Glu Leu Gln Glu Ile Arg Leu 275 280 272 amino acidsamino acid <Unknown> linear 6 Met Ser Pro Ile Leu Gly Tyr Trp Lys IleLys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu GluGlu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys TrpArg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro TyrTyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile ArgTyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys GluArg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg TyrGly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu LysVal Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe GluAsp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val ThrHis Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val LeuTyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val CysPhe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 LeuLys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Pro Arg Glu Phe 210 215220 Ile Val Thr Asp Met Ala Gly Val Ala Cys Leu Gly Lys Thr Ala Asp 225230 235 240 Ala Asp Glu Trp Cys Asp Ala Gly Leu Gly Ala Leu Gly Pro AspAla 245 250 255 Ala Ala Pro Gly Gly Pro Gly Leu Gly Ala Glu Leu Gly ProGlu Leu 260 265 270 2251 base pairs nucleic acid single linear 7GGCACGAGGC CCCATGGAGC GGCCCCCGGG GCTGCGGCCG GGCGCGGGCG GGCCCTGGGA 60GATGCGGGAG CGGCTGGGCA CCGGCGGCTT CGGGAACGTC TGTCTGTACC AGCATCGGGA 120ACTTGATCTC AAAATAGCAA TTAAGTCTTG TCGCCTAGAG CTAAGTACCA AAAACAGAGA 180ACGATGGTGC CATGAAATCC AGATTATGAA GAAGTTGAAC CATGCCAATG TTGTAAAGGC 240CTGTGATGTT CCTGAAGAAT TGAATATTTT GATTCATGAT GTGCCTCTTC TAGCAATGGA 300ATACTGTTCT GGAGGAGATC TCCGAAAGCT GCTCAACAAA CCAGAAAATT GTTGTGGACT 360TAAAGAAAGC CAGATACTTT CTTTACTAAG TGATATAGGG TCTGGGATTC GATATTTGCA 420TGAAAACAAA ATTATACATC GAGATCTAAA ACCTGAAAAC ATAGTTCTTC AGGATGTTGG 480TGGAAAGATA ATACATAAAA TAATTGATCT GGGATATGCC AAAGATGTTG ATCAAGGAAG 540TCTGTGTACA TCTTTTGTGG GAACACTGCA GTATCTGGCC CCAGAGCTCT TTGAGAATAA 600GCCTTACACA GCCACTGTTG ATTATTGGAG CTTTGGGACC ATGGTATTTG AATGTATTGC 660TGGATATAGG CCTTTTTTGC ATCATCTGCA GCCATTTACC TGGCATGAGA AGATTAAGAA 720GAAGGATCCA AAGTGTATAT TTGCATGTGA AGAGATGTCA GGAGAAGTTC GGTTTAGTAG 780CCATTTACCT CAACCAAATA GCCTTTGTAG TTTAATAGTA GAACCCATGG AAAACTGGCT 840ACAGTTGATG TTGAATTGGG ACCCTCAGCA GAGAGGAGGA CCTGTTGACC TTACTTTGAA 900GCAGCCAAGA TGTTTTGTAT TAATGGATCA CATTTTGAAT TTGAAGATAG TACACATCCT 960AAATATGACT TCTGCAAAGA TAATTTCTTT TCTGTTACCA CCTGATGAAA GTCTTCATTC 1020ACTACAGTCT CGTATTGAGC GTGAAACTGG AATAAATACT GGTTCTCAAG AACTTCTTTC 1080AGAGACAGGA ATTTCTCTGG ATCCTCGGAA ACCAGCCTCT CAATGTGTTC TAGATGGAGT 1140TAGAGGCTGT GATAGCTATA TGGTTTATTT GTTTGATAAA AGTAAAACTG TATATGAAGG 1200GCCATTTGCT TCCAGAAGTT TATCTGATTG TGTAAATTAT ATTGTACAGG ACAGCAAAAT 1260ACAGCTTCCA ATTATACAGC TGCGTAAAGT GTGGGCTGAA GCAGTGCACT ATGTGTCTGG 1320ACTAAAAGAA GACTATAGCA GGCTCTTTCA GGGACAAAGG GCAGCAATGT TAAGTCTTCT 1380TAGATATAAT GCTAACTTAA CAAAAATGAA GAACACTTTG ATCTCAGCAT CACAACAACT 1440GAAAGCTAAA TTGGAGTTTT TTCACAAAAG CATTCAGCTT GACTTGGAGA GATACAGCGA 1500GCAGATGACG TATGGGATAT CTTCAGAAAA AATGCTAAAA GCATGGAAAG AAATGGAAGA 1560AAAGGCCATC CACTATGCTG AGGTTGGTGT CATTGGATAC CTGGAGGATC AGATTATGTC 1620TTTGCATGCT GAAATCATGG AGCTACAGAA GAGCCCCTAT GGAAGACGTC AGGGAGACTT 1680GATGGAATCT CTGGAACAGC GTGCCATTGA TCTATATAAG CAGTTAAAAC ACAGACCTTC 1740AGATCACTCC TACAGTGACA GCACAGAGAT GGTGAAAATC ATTGTGCACA CTGTGCAGAG 1800TCAGGACCGT GTGCTCAAGG AGCGTTTTGG TCATTTGAGC AAGTTGTTGG GCTGTAAGCA 1860GAAGATTATT GATCTACTCC CTAAGGTGGA AGTGGCCCTC AGTAATATCA AAGAAGCTGA 1920CAATACTGTC ATGTTCATGC AGGGAAAAAG GCAGAAAGAA ATATGGCATC TCCTTAAAAT 1980TGCCTGTACA CAGAGTTCTG CCCGCTCTCT TGTAGGATCC AGTCTAGAAG GTGCAGTAAC 2040CCCTCAAGCA TACGCATGGC TGGCCCCCGA CTTAGCAGAA CATGATCATT CTCTGTCATG 2100TGTGGTAACT CCTCAAGATG GGGAGACTTC AGCACAAATG ATAGAAGAAA ATTTGAACTG 2160CCTTGGCCAT TTAAGCACTA TTATTCATGA GGCAAATGAG GAACAGGGCA ATAGTATGAT 2220GAATCTTGAT TGGAGTTGGT TAACAGAATG A 2251 2271 base pairs nucleic acidsingle linear 8 ATGAGCTGGT CACCTTCCCT GACAACGCAG ACATGTGGGG CCTGGGAAATGAAAGAGCGC 60 CTTGGGACAG GGGGATTTGG AAATGTCATC CGATGGCACA ATCAGGAAACAGGTGAGCAG 120 ATTGCCATCA AGCAGTGCCG GCAGGAGCTC AGCCCCCGGA ACCGAGAGCGGTGGTGCCTG 180 GAGATCCAGA TCATGAGAAG GCTGACCCAC CCCAATGTGG TGGCTGCCCGAGATGTCCCT 240 GAGGGGATGC AGAACTTGGC GCCCAATGAC CTGCCCCTGC TGGCCATGGAGTACTGCCAA 300 GGAGGAGATC TCCGGAAGTA CCTGAACCAG TTTGAGAACT GCTGTGGTCTGCGGGAAGGT 360 GCCATCCTCA CCTTGCTGAG TGACATTGCC TCTGCGCTTA GATACCTTCATGAAAACAGA 420 ATCATCCATC GGGATCTAAA GCCAGAAAAC ATCGTCCTGC AGCAAGGAGAACAGAGGTTA 480 ATACACAAAA TTATTGACCT AGGATATGCC AAGGAGCTGG ATCAGGGCAGTCTTTGCACA 540 TCATTCGTGG GGACCCTGCA GTACCTGGCC CCAGAGCTAC TGGAGCAGCAGAAGTACACA 600 GTGACCGTCG ACTACTGGAG CTTCGGCACC CTGGCCTTTG AGTGCATCACGGGCTTCCGG 660 CCCTTCCTCC CCAACTGGCA GCCCGTGCAG TGGCATTCAA AAGTGCGGCAGAAGAGTGAG 720 GTGGACATTG TTGTTAGCGA AGACTTGAAT GGAACGGTGA AGTTTTCAAGCTCTTTACCC 780 TACCCCAATA ATCTTAACAG TGTCCTGGCT GAGCGACTGG AGAAGTGGCTGCAACTGATG 840 CTGATGTGGC ACCCCCGACA GAGGGGCACG GATCCCACGT ATGGGCCCAATGGCTGCTTC 900 AAGGCCCTGG ATGACATCTT AAACTTAAAG TTGGTTCATA TCTTGAACATGGTCACGGGC 960 ACCATCCACA CCTACCCTGT GACAGAGGAT GAGAGTCTGC AGAGCTTGAAGGCCAGAATC 1020 CAACAGGACA CGGGCATCCC AGAGGAGGAC CAGGAGCTGC TGCAGGAAGCGGGCCTGGCG 1080 TTGATCCCCG ATAAGCCTGC CACTCAGTGT ATTTCAGACG GCAAGTTAAATGAGGGCCAC 1140 ACATTGGACA TGGATCTTGT TTTTCTCTTT GACAACAGTA AAATCACCTATGAGACTCAG 1200 ATCTCCCCAC GGCCCCAACC TGAAAGTGTC AGCTGTATCC TTCAAGAGCCCAAGAGGAAT 1260 CTCGCCTTCT TCCACCTGAG GAAGGTGTGG GGCCAGGTCT GGCACAGCATCCAGACCCTG 1320 AAGGAAGATT GCAACCGGCT GCAGCAGGGA CAGCGAGCCG CCATGATGAATCTCCTCCGA 1380 AACAACAGCT GCCTCTCCAA AATGAAGAAT TCCATGGCTT CCATGTCTCAGCAGCTCAAG 1440 GCCAAGTTGG ATTTCTTCAA AACCAGCATC CAGATTGACC TGGAGAAGTACAGCGAGCAA 1500 ACCGAGTTTG GGATCACATC AGATAAACTG CTGCTGGCCT GGAGGGAAATGGAGCAGGCT 1560 GTGGAGCTCT GTGGGCGGGA GAACGAAGTG AAACTCCTGG TAGAACGGATGATGGCTCTG 1620 CAGACCGACA TTGTGGACTT ACAGAGGAGC CCCATGGGCC GGAAGCAGGGGGGAACGCTG 1680 GACGACCTAG AGGAGCAAGC AAGGGAGCTG TACAGGAGAC TAAGGGAAAAACCTCGAGAC 1740 CAGCGAACTG AGGGTGACAG TCAGGAAATG GTACGGCTGC TGCTTCAGGCAATTCAGAGC 1800 TTCGAGAAGA AAGTGCGAGT GATCTATACG CAGCTCAGTA AAACTGTGGTTTGCAAGCAG 1860 AAGGCGCTGG AACTGTTGCC CAAGGTGGAA GAGGTGGTGA GCTTAATGAATGAGGATGAG 1920 AAGACTGTTG TCCGGCTGCA GGAGAAGCGG CAGAAGGAGC TCTGGAATCTCCTGAAGATT 1980 GCTTGTAGCA AGGTCCGTGG TCCTGTCAGT GGAAGCCCGG ATAGCATGAATGCCTCTCGA 2040 CTTAGCCAGC CTGGGCAGCT GATGTCTCAG CCCTCCACGG CCTCCAACAGCTTACCTGAG 2100 CCAGCCAAGA AGAGTGAAGA ACTGGTGGCT GAAGCACATA ACCTCTGCACCCTGCTAGAA 2160 AATGCCATAC AGGACACTGT GAGGGAACAA GACCAGAGTT TCACGGCCCTAGACTGGAGC 2220 TGGTTACAGA CGGAAGAAGA AGAGCACAGC TGCCTGGAGC AGGCCTCATG A2271 756 amino acids amino acid <Unknown> linear 9 Met Ser Trp Ser ProSer Leu Thr Thr Gln Thr Cys Gly Ala Trp Glu 1 5 10 15 Met Lys Glu ArgLeu Gly Thr Gly Gly Phe Gly Asn Val Ile Arg Trp 20 25 30 His Asn Gln GluThr Gly Glu Gln Ile Ala Ile Lys Gln Cys Arg Gln 35 40 45 Glu Leu Ser ProArg Asn Arg Glu Arg Trp Cys Leu Glu Ile Gln Ile 50 55 60 Met Arg Arg LeuThr His Pro Asn Val Val Ala Ala Arg Asp Val Pro 65 70 75 80 Glu Gly MetGln Asn Leu Ala Pro Asn Asp Leu Pro Leu Leu Ala Met 85 90 95 Glu Tyr CysGln Gly Gly Asp Leu Arg Lys Tyr Leu Asn Gln Phe Glu 100 105 110 Asn CysCys Gly Leu Arg Glu Gly Ala Ile Leu Thr Leu Leu Ser Asp 115 120 125 IleAla Ser Ala Leu Arg Tyr Leu His Glu Asn Arg Ile Ile His Arg 130 135 140Asp Leu Lys Pro Glu Asn Ile Val Leu Gln Gln Gly Glu Gln Arg Leu 145 150155 160 Ile His Lys Ile Ile Asp Leu Gly Tyr Ala Lys Glu Leu Asp Gln Gly165 170 175 Ser Leu Cys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala ProGlu 180 185 190 Leu Leu Glu Gln Gln Lys Tyr Thr Val Thr Val Asp Tyr TrpSer Phe 195 200 205 Gly Thr Leu Ala Phe Glu Cys Ile Thr Gly Phe Arg ProPhe Leu Pro 210 215 220 Asn Trp Gln Pro Val Gln Trp His Ser Lys Val ArgGln Lys Ser Glu 225 230 235 240 Val Asp Ile Val Val Ser Glu Asp Leu AsnGly Thr Val Lys Phe Ser 245 250 255 Ser Ser Leu Pro Tyr Pro Asn Asn LeuAsn Ser Val Leu Ala Glu Arg 260 265 270 Leu Glu Lys Trp Leu Gln Leu MetLeu Met Trp His Pro Arg Gln Arg 275 280 285 Gly Thr Asp Pro Thr Tyr GlyPro Asn Gly Cys Phe Lys Ala Leu Asp 290 295 300 Asp Ile Leu Asn Leu LysLeu Val His Ile Leu Asn Met Val Thr Gly 305 310 315 320 Thr Ile His ThrTyr Pro Val Thr Glu Asp Glu Ser Leu Gln Ser Leu 325 330 335 Lys Ala ArgIle Gln Gln Asp Thr Gly Ile Pro Glu Glu Asp Gln Glu 340 345 350 Leu LeuGln Glu Ala Gly Leu Ala Leu Ile Pro Asp Lys Pro Ala Thr 355 360 365 GlnCys Ile Ser Asp Gly Lys Leu Asn Glu Gly His Thr Leu Asp Met 370 375 380Asp Leu Val Phe Leu Phe Asp Asn Ser Lys Ile Thr Tyr Glu Thr Gln 385 390395 400 Ile Ser Pro Arg Pro Gln Pro Glu Ser Val Ser Cys Ile Leu Gln Glu405 410 415 Pro Lys Arg Asn Leu Ala Phe Phe His Leu Arg Lys Val Trp GlyGln 420 425 430 Val Trp His Ser Ile Gln Thr Leu Lys Glu Asp Cys Asn ArgLeu Gln 435 440 445 Gln Gly Gln Arg Ala Ala Met Met Asn Leu Leu Arg AsnAsn Ser Cys 450 455 460 Leu Ser Lys Met Lys Asn Ser Met Ala Ser Met SerGln Gln Leu Lys 465 470 475 480 Ala Lys Leu Asp Phe Phe Lys Thr Ser IleGln Ile Asp Leu Glu Lys 485 490 495 Tyr Ser Glu Gln Thr Glu Phe Gly IleThr Ser Asp Lys Leu Leu Leu 500 505 510 Ala Trp Arg Glu Met Glu Gln AlaVal Glu Leu Cys Gly Arg Glu Asn 515 520 525 Glu Val Lys Leu Leu Val GluArg Met Met Ala Leu Gln Thr Asp Ile 530 535 540 Val Asp Leu Gln Arg SerPro Met Gly Arg Lys Gln Gly Gly Thr Leu 545 550 555 560 Asp Asp Leu GluGlu Gln Ala Arg Glu Leu Tyr Arg Arg Leu Arg Glu 565 570 575 Lys Pro ArgAsp Gln Arg Thr Glu Gly Asp Ser Gln Glu Met Val Arg 580 585 590 Leu LeuLeu Gln Ala Ile Gln Ser Phe Glu Lys Lys Val Arg Val Ile 595 600 605 TyrThr Gln Leu Ser Lys Thr Val Val Cys Lys Gln Lys Ala Leu Glu 610 615 620Leu Leu Pro Lys Val Glu Glu Val Val Ser Leu Met Asn Glu Asp Glu 625 630635 640 Lys Thr Val Val Arg Leu Gln Glu Lys Arg Gln Lys Glu Leu Trp Asn645 650 655 Leu Leu Lys Ile Ala Cys Ser Lys Val Arg Gly Pro Val Ser GlySer 660 665 670 Pro Asp Ser Met Asn Ala Ser Arg Leu Ser Gln Pro Gly GlnLeu Met 675 680 685 Ser Gln Pro Ser Thr Ala Ser Asn Ser Leu Pro Glu ProAla Lys Lys 690 695 700 Ser Glu Glu Leu Val Ala Glu Ala His Asn Leu CysThr Leu Leu Glu 705 710 715 720 Asn Ala Ile Gln Asp Thr Val Arg Glu GlnAsp Gln Ser Phe Thr Ala 725 730 735 Leu Asp Trp Ser Trp Leu Gln Thr GluGlu Glu Glu His Ser Cys Leu 740 745 750 Glu Gln Ala Ser 755 745 aminoacids amino acid <Unknown> linear 10 Met Glu Arg Pro Pro Gly Leu Arg ProGly Ala Gly Gly Pro Trp Glu 1 5 10 15 Met Arg Glu Arg Leu Gly Thr GlyGly Phe Gly Asn Val Cys Leu Tyr 20 25 30 Gln His Arg Glu Leu Asp Leu LysIle Ala Ile Lys Ser Cys Arg Leu 35 40 45 Glu Leu Ser Thr Lys Asn Arg GluArg Trp Cys His Glu Ile Gln Ile 50 55 60 Met Lys Lys Leu Asn His Ala AsnVal Val Lys Ala Cys Asp Val Pro 65 70 75 80 Glu Glu Leu Asn Ile Leu IleHis Asp Val Pro Leu Leu Ala Met Glu 85 90 95 Tyr Cys Ser Gly Gly Asp LeuArg Lys Leu Leu Asn Lys Pro Glu Asn 100 105 110 Cys Cys Gly Leu Lys GluSer Gln Ile Leu Ser Leu Leu Ser Asp Ile 115 120 125 Gly Ser Gly Ile ArgTyr Leu His Glu Asn Lys Ile Ile His Arg Asp 130 135 140 Leu Lys Pro GluAsn Ile Val Leu Gln Asp Val Gly Gly Lys Ile Ile 145 150 155 160 His LysIle Ile Asp Leu Gly Tyr Ala Lys Asp Val Asp Gln Gly Ser 165 170 175 LeuCys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu Leu 180 185 190Phe Glu Asn Lys Pro Tyr Thr Ala Thr Val Asp Tyr Trp Ser Phe Gly 195 200205 Thr Met Val Phe Glu Cys Ile Ala Gly Tyr Arg Pro Phe Leu His His 210215 220 Leu Gln Pro Phe Thr Trp His Glu Lys Ile Lys Lys Lys Asp Pro Lys225 230 235 240 Cys Ile Phe Ala Cys Glu Glu Met Ser Gly Glu Val Arg PheSer Ser 245 250 255 His Leu Pro Gln Pro Asn Ser Leu Cys Ser Leu Ile ValGlu Pro Met 260 265 270 Glu Asn Trp Leu Gln Leu Met Leu Asn Trp Asp ProGln Gln Arg Gly 275 280 285 Gly Pro Val Asp Leu Thr Leu Lys Gln Pro ArgCys Phe Val Leu Met 290 295 300 Asp His Ile Leu Asn Leu Lys Ile Val HisIle Leu Asn Met Thr Ser 305 310 315 320 Ala Lys Ile Ile Ser Phe Leu LeuPro Pro Asp Glu Ser Leu His Ser 325 330 335 Leu Gln Ser Arg Ile Glu ArgGlu Thr Gly Ile Asn Thr Gly Ser Gln 340 345 350 Glu Leu Leu Ser Glu ThrGly Ile Ser Leu Asp Pro Arg Lys Pro Ala 355 360 365 Ser Gln Cys Val LeuAsp Gly Val Arg Gly Cys Asp Ser Tyr Met Val 370 375 380 Tyr Leu Phe AspLys Ser Lys Thr Val Tyr Glu Gly Pro Phe Ala Ser 385 390 395 400 Arg SerLeu Ser Asp Cys Val Asn Tyr Ile Val Gln Asp Ser Lys Ile 405 410 415 GlnLeu Pro Ile Ile Gln Leu Arg Lys Val Trp Ala Glu Ala Val His 420 425 430Tyr Val Ser Gly Leu Lys Glu Asp Tyr Ser Arg Leu Phe Gln Gly Gln 435 440445 Arg Ala Ala Met Leu Ser Leu Leu Arg Tyr Asn Ala Asn Leu Thr Lys 450455 460 Met Lys Asn Thr Leu Ile Ser Ala Ser Gln Gln Leu Lys Ala Lys Leu465 470 475 480 Glu Phe Phe His Lys Ser Ile Gln Leu Asp Leu Glu Arg TyrSer Glu 485 490 495 Gln Met Thr Tyr Gly Ile Ser Ser Glu Lys Met Leu LysAla Trp Lys 500 505 510 Glu Met Glu Glu Lys Ala Ile His Tyr Ala Glu ValGly Val Ile Gly 515 520 525 Tyr Leu Glu Asp Gln Ile Met Ser Leu His AlaGlu Ile Met Glu Leu 530 535 540 Gln Lys Ser Pro Tyr Gly Arg Arg Gln GlyAsp Leu Met Glu Ser Leu 545 550 555 560 Glu Gln Arg Ala Ile Asp Leu TyrLys Gln Leu Lys His Arg Pro Ser 565 570 575 Asp His Ser Tyr Ser Asp SerThr Glu Met Val Lys Ile Ile Val His 580 585 590 Thr Val Gln Ser Gln AspArg Val Leu Lys Glu Arg Phe Gly His Leu 595 600 605 Ser Lys Leu Leu GlyCys Lys Gln Lys Ile Ile Asp Leu Leu Pro Lys 610 615 620 Val Glu Val AlaLeu Ser Asn Ile Lys Glu Ala Asp Asn Thr Val Met 625 630 635 640 Phe MetGln Gly Lys Arg Gln Lys Glu Ile Trp His Leu Leu Lys Ile 645 650 655 AlaCys Thr Gln Ser Ser Ala Arg Ser Leu Val Gly Ser Ser Leu Glu 660 665 670Gly Ala Val Thr Pro Gln Ala Tyr Ala Trp Leu Ala Pro Asp Leu Ala 675 680685 Glu His Asp His Ser Leu Ser Cys Val Val Thr Pro Gln Asp Gly Glu 690695 700 Thr Ser Ala Gln Met Ile Glu Glu Asn Leu Asn Cys Leu Gly His Leu705 710 715 720 Ser Thr Ile Ile His Glu Ala Asn Glu Glu Gln Gly Asn SerMet Met 725 730 735 Asn Leu Asp Trp Ser Trp Leu Thr Glu 740 745 22 aminoacids amino acid <Unknown> linear 11 Cys Lys Lys Glu Arg Leu Leu Asp AspArg His Asp Ser Gly Leu Asp 1 5 10 15 Ser Met Lys Asp Glu Glu 20 22amino acids amino acid <Unknown> linear 12 Cys Lys Lys Glu Arg Leu LeuAsp Asp Arg His Asp Thr Gly Leu Asp 1 5 10 15 Thr Met Lys Asp Glu Glu 2019 amino acids amino acid <Unknown> linear Modified-site 10 /note=“Where Xaa is a Phosphate Ester of Threonine” 13 Asp Leu Thr Gly Gly ProGlu Val Ala Xaa Pro Glu Ser Glu Glu Ala 1 5 10 15 Phe Leu Pro 19 aminoacids amino acid <Unknown> linear 14 Cys Pro Thr Asn Ser Ala Leu Asn TyrLeu Lys Ser Pro Ile Thr Thr 1 5 10 15 Ser Pro Ser 15 amino acids aminoacid <Unknown> linear 15 Cys Asn Ser Asp Leu Leu Thr Ser Pro Asp Val GlyLeu Leu Lys 1 5 10 15 15 amino acids amino acid <Unknown> linear 16 CysVal Gly Leu Leu Lys Leu Ala Ser Pro Glu Leu Glu Arg Leu 1 5 10 15 8amino acids amino acid <Unknown> linear 17 Ile Ile Asp Leu Gly Tyr AlaLys 1 5 9 amino acids amino acid <Unknown> linear 18 Val Glu Val Ala LeuSer Asn Ile Lys 1 5 8 amino acids amino acid <Unknown> linear 19 Ser IleGln Leu Asp Leu Glu Arg 1 5 7 amino acids amino acid <Unknown> linear 20Ala Leu Glu Leu Leu Pro Lys 1 5 8 amino acids amino acid <Unknown>linear 21 Val Ile Tyr Thr Gln Leu Ser Lys 1 5 11 amino acids amino acid<Unknown> linear 22 Leu Leu Leu Gln Ala Ile Gln Ser Phe Glu Lys 1 5 1011 amino acids amino acid <Unknown> linear 23 Leu Gly Thr Gly Gly PheGly Asn Val Ile Arg 1 5 10 9 amino acids amino acid <Unknown> linear 24Ala Leu Asp Asp Ile Leu Asn Leu Lys 1 5 15 amino acids amino acid<Unknown> linear 25 Asp Leu Lys Pro Glu Asn Ile Val Leu Gln Gln Gly GluGln Arg 1 5 10 15

1. An IKK signalsome capable of specifically phosphorylating IκBα atresidues S32 and S36, and IκBβ at residues 19 and 23, without theaddition of exogenous cofactors.
 2. An IKK signalsome according to claim1 wherein the signalsome is derived from a human tissue or cell line. 3.A polypeptide comprising a component of an IKK signalsome according toclaim 1, or a variant of such a component, wherein the component has asequence recited in SEQ ID NO:9.
 4. An isolated DNA molecule encoding apolypeptide according to claim
 3. 5. A recombinant expression vectorcomprising a DNA molecule according to claim
 4. 6. A host celltransformed or transfected with an expression vector according to claim5.
 7. A host cell according to claim 6, wherein the host cell isselected from the group consisting of bacteria, yeast, baculovirusinfected insect cells and mammalian cells.
 8. A method for preparing anIKK signalsome, comprising combining components of an IKK signalsome ina suitable buffer.
 9. A method for phosphorylating a substrate of an IKKsignalsome, comprising contacting a substrate with a signalsomeaccording to claim 1 and thereby phosphorylating the substrate.
 10. Amethod for phosphorylating a substrate of an IKK signalsome, comprisingcontacting a substrate with a polypeptide comprising a component of anIKK signalsome having IκB kinase activity, and thereby phosphorylatingthe substrate.
 11. A method according to claim 10, wherein thepolypeptide comprises IKK-1 (SEQ ID NO:10).
 12. A method according toclaim 10, wherein the polypeptide comprises IKK-2 (SEQ ID NO:9).
 13. Themethod of either of claims 9 or 10, wherein the substrate is IκBα or avariant thereof.
 14. A method for screening for an agent that modulatesIKK signalsome activity, comprising: (a) contacting a candidate agentwith an IKK signalsome according to claim 1, wherein the step ofcontacting is carried out under conditions and for a time sufficient toallow the candidate agent and the IKK signalsome to interact; and (b)subsequently measuring the ability of the candidate agent to modulate aIKK signalsome activity.
 15. A method according to claim 14, wherein theIKK signalsome activity modulated is selected from the group consistingof IκB kinase activity, p65 kinase activity and IKK phosphataseactivity.
 16. A method for screening for an agent that modulates IKKsignalsome activity, comprising: (a) contacting a candidate agent with apolypeptide comprising a component of an IKK signalsome according toclaim 1, wherein the step of contacting is carried out under conditionsand for a time sufficient to allow the candidate agent and thepolypeptide to interact; and (b) subsequently measuring the ability ofthe candidate agent to modulate the ability of the polypeptide tophosphorylate an IκB protein.
 17. A method according to claim 16,wherein the polypeptide comprises IKK-1 (SEQ ID NO:10).
 18. A methodaccording to claim 16, wherein the polypeptide comprises IKK-2 (SEQ IDNO:9).
 19. An antibody that binds to IKK-1 (SEQ ID NO:10). and/or IKK-2(SEQ ID NO:9).
 20. An antibody according to claim 19, wherein theantibody inhibits the phosphorylation of an IκB protein by an IKKsignalsome.
 21. A method for modulating NF-κB activity in a patient,comprising administering to a patient an agent that modulates IKKsignalsome activity in combination with a pharmaceutically acceptablecarrier, and thereby modulating NF-κB activity in the patient.
 22. Themethod of claim 21, wherein the agent inhibits activation of an IKKsignalsome.
 23. The method of claim 21, wherein the agentinhibits-kinase activity of an activated IKK signalsome.
 24. A methodfor treating a patient afflicted with a disorder associated with theactivation of an IKK signalsome, comprising administering to a patient atherapeutically effective amount of an agent that modulates IKKsignalsome activity in combination with a pharmaceutically acceptablecarrier.
 25. The method of any one of claims 21-24, wherein the agent isa monoclonal antibody.
 26. The method of any of claims 21-24, whereinthe agent comprises a polynucleotide.
 27. A method for detecting IKKsignalsome activity in a sample, comprising: (a) contacting a sample:with an antibody that binds to an IKK signalsome under conditions andfor a time sufficient to allow the antibody to immunoprecipitate an IKKsignalsome; (b) separating immunoprecipitated material from the sample;and (c) determining the ability of the immunoprecipitated material tophosphorylate an IκB protein with in vivo specificity.
 28. A methodaccording to claim 27, wherein the immunoprecipitated materialphosphorylates IκBα at residues S32 and S36.
 29. A kit for detecting IKKsignalsome activity in a sample, comprising an antibody that binds to aIKK signalsome in combination with a suitable buffer.
 30. A method foridentifying an upstream kinase in the NF-κB signal transduction cascade,comprising evaluating the ability of a candidate upstream kinase tophosphorylate and induce enzymatic activity of an IKK signalsome or acomponent or variant thereof, and thereby identifying an upstream kinasein the NF-κB signal transduction cascade.
 31. A method for identifying acomponent of an IKK signalsome, comprising: (a) isolating an IKKsignalsome; (b) separating the signalsome into components; and (c)obtaining a partial sequence of a component, and thereby identifying acomponent of an IKK signalsome.
 32. A method for preparing an IKKsignalsome from a biological sample, comprising: (a) separating abiological sample into two or more fractions; and (b) monitoring IκBkinase activity in the fractions.